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The stereochemistry of cyclization of the bacterial C50Carotenoid C.p.450
Pergamoa
Phyrahcmurry,
Vol. 35. No. 4. pp. 931 -934. 1994 Elscvm Scieocc Ltd PfioIcd In Grul Bntain. 003
I 9mp4s6.a) +0.00
THE STEREOCHEMISTRY OF CYCLIZATION OF THE BACTERIAL C&AROTENOID C.p.450 S.H.
RHODES* and 9. V. MILBORROW
School of Biochemistry and Molecular Genetics, University of New South Wales, P.O. Box I, Kensington, N.S.W. 2033, Australia (Received 23 April 1993) Key Word Index-Carotenoid;
C.p.450; cyclization;
NMR; stereochemistry.
Abstract-The ‘H and i3CNMR signals of the uplield, C-l methyl group of the C,, bacterial carotenoid, C.p.450, have been identified as those of the equatorial, C-l-pro-R methyl group by ‘H-i% correlation, decoupling and NOE experiments. This reverses the previous, tentative assignment (Britton. 1985, citing results of Englert) and thereby reestablishes the possibility of chair-folding of the precursor during cyclization.
INTRODUCIION
In the biosynthesis of cyclic carotenoids the final disposition of the various ring substituents at C-l, C-2 and C-6 is defined by two features of the cyclization reaction. These are, firstly, the face from which the electrophilic attack occurs at C-2 (by a proton in the case of C.+ccarotenoids and a C, moiety for the C,, series), and secondly, the conformation of the folded precursor [l]. The stereochemistry of cyclization for the C,, b,/?carotenoid zeaxanthin (1) in a Flaoobacterium sp. has been elucidated by Britton et al. [2, 33. Biosynthetic labelling with deuterium at C-2 demonstrated that the H atom, initially from the medium, added during cyclization, was present in the C-2-pro-R position, i.e. on the /Iface, upper as usually drawn. Mevalonate enriched with 13C at C-2 and incorporated into zeaxanthin enhanced the i3C signal of the upfield Me of the geminul pair at C-l in the NMR spectrum. Detailed analysis of the NMR spectrum [4] assigned this as the signal of the axial, a, C-l -pro-S methyl carbon. In the ‘HNMR spectrum, however, this Me gave rise to the downfield signal of the C-l geminal pair of methyl groups. This was further confirmed by selective enrichment of the axial Me with deuterium when the organism was grown in C2H]H20 on glucose [S]. The lutein (2) formed by an alga grown in C2H]H,0 also gave C-1’-pro-S-Me and C2’-pro-R-H enriched with 2H (in the s-ring), so the stereochemistry of attack by H’ was shown to be the same for both /I- and c-ring (Fig. 1) [6]. This arrangement of the C-l methyl groups in the s-ring was shown to be the same as for the @ing of both carotenoids by 13C-13C coupling experiments [5]. Enhancement of the upfield C-l methyl signal has also been reported [S] for the C,c carotenoid C.p.450 (3) *Present address: Minenco Pty., Level 5, 77 Berry Street. North Sydney 2060, NSW, Australia. PHYTO 35: 4-H
HO-
[4]
Dceaprenoxamhin (t. l)
‘2
synthesized by Corynebacterium poinsettiae cultured in the presence of [2-13C]mevalonate. This signal was tentatively identified as that of the C-l, axial, C-l-pro-S methyl carbon. Further, selective deuterium enrichment during growth of Cellulomonas (previously Flaoobacterium) dehydrogenans on glucose in 2H20 yielded decaprenoxanthin (the E,E-CsOanalogue of C.p.450) [4] in which the higher field ‘H signal, assigned to the axial, pro-S Me, showed 2H enrichment, and originated, therefore, from C-2 of mevalonate. It was concluded by Britton [S] that
931
S. H.
932
RHODESand B. V. MILBORROW
We have determined the absolute configuration of the C-l geminal methyl groups of C.p.450 by using the ‘H signals to relate their stereochemistry to that at C-2 whose absolute configuration has been established [8]. The ‘H signals of the C-l Me groups were then related to the *jC signals by a 13C-‘H correlation experiment. Then, by using the biosynthetic data given by B&ton [S], we have deduced the stereochemistry of the precursor during cyclization. RESULTS
Fig. I. Stereochemistry of attack by H+.
Table 1. ‘HNMR spectral data for C.p.450 (500 MHz. [2H,]benzene)
C 1Me IMe
2 3 3eq 4 1” 2” 4” 5”Me
Chemical shift 6 (ppm)
Coupling constants J (Hz)
1.05
1.21 1.42 1.41 1.77 - 2.00
1.86 2.26 5.45 3.83 1.61
11.2.7, 1.5 14,6.0,4 14,6.0 6.0, 6.0
Data for the ring and the C-2 side chain are shown.
the same stereochemistry occurred at C-l in these C,, carotenoids as that at C-l in the C,, series. In contrast to C-l, the H atom and the isopentenyl residue added at C-2, and the absolute configuration of the H atom at C-6 of decaprenoxanthin were in the mirror-image positions compared with their counterparts in the C,, carotenoids. The consequences of the tentative stereochemistry assigned at C-l of C.p.450 is that the postulated cyclization mechanism is not consistent with the observed stereochemistry at C-2 and C-6 of the C,,, carotenoids, and a more complicated reaction mechanism is required than had been previously proposed [ 1J. The conformation of a molecule is usually more readily determined from the ‘H than from the “C spectrum but care has to be exercised to establish the precise identity of groups that give rise to particular “C and ‘H signals. For example, the homologous geminal Me signals of the phytohormone abscisic acid reverse their relative chemical shifts between ‘H and 13C spectra [7].
Chemical shifts obtained in [*H,J benzene are given in Table 1. The signal observed to be enriched by incorporation of [2-“C] mevalonate was that at 22.54 ppm in [*HI chloroform (Englert, unpublished results cited by B&ton [SJ) and was identified as that at 22.71 ppm in [*Ha] benzene, its downfield partner being found at 28.4 ppm (chloroform) and 27.64 ppm (benzene). A 13C-*H correlation experiment showed that the relative positions of the geminal C-l methyl signals were the same in both spectra. The upfield signals in the proton spectrum (1.05 ppm) correlated with those in the “C spectrum (22.71 ppm), and likewise for the downfield signals (1.21 ppm in the ‘H spectrum, correlated with the signal at 27.64 ppm in the ‘“C spectrum). The proton signal from C-2 was identified at 1.42 ppm (45.8 ppm in t3C spectra). The protons at C-l” were found to give separate signals at 1.86 and 2.26 ppm, thereby indicating that there was very restricted rotation about the C-2-Cl” bond. Correlated 20 spectroscopy (COS Y)
Analysis of the COSY spectrum revealed geminul coup lings between the two C-l” protons (1.86 and 2.26 ppm). The proton at C-2 was coupled to only one of the protons at C-l” (1.86 ppm), and coupling was seen between the C2 and one of the C-3 protons (1.77 ppm). Coupling between the C-3 protons (1.77 and 1.41 ppm) and between the geminal C-3 protons was also observed, and between the C-3 protons (1.77 ppm and 1.41 ppm) and the protons at C-4 (2.00 ppm). In addition, coupling between the Me at C-5 (1.81 ppm) was observed to the C-4 signal. NOE experiments
A ‘H NOE by difference experiment gave several small enhancements when either of the geminal-Me-l signals were irradiated, in particular those at 6.35 ppm (protons at C-7, C-8), and the C-l” H signal at 2.26 ppm, but not that of the other C-l” H at 1.86 ppm. Also, when the upfield Me was irradiated, a very small enhancement of the signal at 1.42 ppm was observed; when the downfield Me was irradiated small enhancements were observed for the signals at 1.61 ppm (C5” methyl) and at 1.77 ppm (C-3). Irradiation at 1.42 ppm, however, gave a large enhancement of the methyl signal at 1.05 ppm, i.e. the
S. H. RHODES and B. V MILB~RROW
934 REFERENCES
1. Eugster, C. H. (1979) Pure Appl. Chem. 51,463. 2. Britton, G., Lockely, W. J. S., Patel, N. J., Goodwin, T. W. and Englert, G. (1977) J. Chem. Sot. Chem. Commun. 655. 3. Britton, G., Goodwin, T. W., Lockley, W. J. S., Mundy, A. P., Pate], N. J. and Englert, G. (1979) J. Chem. Sot. Chem. Commun. 27. 4. Englert, G. (1982) in Carotenoid Chemistry and Eiochemistry (Britton, G. and Goodwin, T. W., eds), p. 107. Pergamon Press, Oxford. 5. Britton, G. (1985) Pure Appl. Chem. 57, 701. 6. Britton, G. and Mundy, A. P. (1980) Dev. Plant Biol. 6, 345.
7. Milborrow, B. V. (1984) Biochem. J 220, 325. S. and Weeks, 0. B. 8. Andrewes, A. C., Liaaen-Jensen, (1975) Acta Chem. Scand. 29, 884. 9. Milborrow, B. V. (1983) in Biosynthesis ofIsoprenoid Compounds (Porter, J. W. and Spurgeon, S. L., eds), Vol. 2, pp. 413-436. John Wiley, New York. Starr, M. P. and Sapirstein, S. (1953) Arch. Biochem. lo. Biophys. 43, 157. S. (1970) 11. Norgard, S., Aasen, A. J. and Liaaen-Jenson, Acta Chem. Scand. 24, 2183. 12. Britton, G. (1986) Meths in Enzymol. 111, 113.
Phyrahcmurry,
Vol. 35. No. 4. pp. 931 -934. 1994 Elscvm Scieocc Ltd PfioIcd In Grul Bntain. 003
I 9mp4s6.a) +0.00
THE STEREOCHEMISTRY OF CYCLIZATION OF THE BACTERIAL C&AROTENOID C.p.450 S.H.
RHODES* and 9. V. MILBORROW
School of Biochemistry and Molecular Genetics, University of New South Wales, P.O. Box I, Kensington, N.S.W. 2033, Australia (Received 23 April 1993) Key Word Index-Carotenoid;
C.p.450; cyclization;
NMR; stereochemistry.
Abstract-The ‘H and i3CNMR signals of the uplield, C-l methyl group of the C,, bacterial carotenoid, C.p.450, have been identified as those of the equatorial, C-l-pro-R methyl group by ‘H-i% correlation, decoupling and NOE experiments. This reverses the previous, tentative assignment (Britton. 1985, citing results of Englert) and thereby reestablishes the possibility of chair-folding of the precursor during cyclization.
INTRODUCIION
In the biosynthesis of cyclic carotenoids the final disposition of the various ring substituents at C-l, C-2 and C-6 is defined by two features of the cyclization reaction. These are, firstly, the face from which the electrophilic attack occurs at C-2 (by a proton in the case of C.+ccarotenoids and a C, moiety for the C,, series), and secondly, the conformation of the folded precursor [l]. The stereochemistry of cyclization for the C,, b,/?carotenoid zeaxanthin (1) in a Flaoobacterium sp. has been elucidated by Britton et al. [2, 33. Biosynthetic labelling with deuterium at C-2 demonstrated that the H atom, initially from the medium, added during cyclization, was present in the C-2-pro-R position, i.e. on the /Iface, upper as usually drawn. Mevalonate enriched with 13C at C-2 and incorporated into zeaxanthin enhanced the i3C signal of the upfield Me of the geminul pair at C-l in the NMR spectrum. Detailed analysis of the NMR spectrum [4] assigned this as the signal of the axial, a, C-l -pro-S methyl carbon. In the ‘HNMR spectrum, however, this Me gave rise to the downfield signal of the C-l geminal pair of methyl groups. This was further confirmed by selective enrichment of the axial Me with deuterium when the organism was grown in C2H]H20 on glucose [S]. The lutein (2) formed by an alga grown in C2H]H,0 also gave C-1’-pro-S-Me and C2’-pro-R-H enriched with 2H (in the s-ring), so the stereochemistry of attack by H’ was shown to be the same for both /I- and c-ring (Fig. 1) [6]. This arrangement of the C-l methyl groups in the s-ring was shown to be the same as for the @ing of both carotenoids by 13C-13C coupling experiments [5]. Enhancement of the upfield C-l methyl signal has also been reported [S] for the C,c carotenoid C.p.450 (3) *Present address: Minenco Pty., Level 5, 77 Berry Street. North Sydney 2060, NSW, Australia. PHYTO 35: 4-H
HO-
[4]
Dceaprenoxamhin (t. l)
‘2
synthesized by Corynebacterium poinsettiae cultured in the presence of [2-13C]mevalonate. This signal was tentatively identified as that of the C-l, axial, C-l-pro-S methyl carbon. Further, selective deuterium enrichment during growth of Cellulomonas (previously Flaoobacterium) dehydrogenans on glucose in 2H20 yielded decaprenoxanthin (the E,E-CsOanalogue of C.p.450) [4] in which the higher field ‘H signal, assigned to the axial, pro-S Me, showed 2H enrichment, and originated, therefore, from C-2 of mevalonate. It was concluded by Britton [S] that
931
S. H.
932
RHODESand B. V. MILBORROW
We have determined the absolute configuration of the C-l geminal methyl groups of C.p.450 by using the ‘H signals to relate their stereochemistry to that at C-2 whose absolute configuration has been established [8]. The ‘H signals of the C-l Me groups were then related to the *jC signals by a 13C-‘H correlation experiment. Then, by using the biosynthetic data given by B&ton [S], we have deduced the stereochemistry of the precursor during cyclization. RESULTS
Fig. I. Stereochemistry of attack by H+.
Table 1. ‘HNMR spectral data for C.p.450 (500 MHz. [2H,]benzene)
C 1Me IMe
2 3 3eq 4 1” 2” 4” 5”Me
Chemical shift 6 (ppm)
Coupling constants J (Hz)
1.05
1.21 1.42 1.41 1.77 - 2.00
1.86 2.26 5.45 3.83 1.61
11.2.7, 1.5 14,6.0,4 14,6.0 6.0, 6.0
Data for the ring and the C-2 side chain are shown.
the same stereochemistry occurred at C-l in these C,, carotenoids as that at C-l in the C,, series. In contrast to C-l, the H atom and the isopentenyl residue added at C-2, and the absolute configuration of the H atom at C-6 of decaprenoxanthin were in the mirror-image positions compared with their counterparts in the C,, carotenoids. The consequences of the tentative stereochemistry assigned at C-l of C.p.450 is that the postulated cyclization mechanism is not consistent with the observed stereochemistry at C-2 and C-6 of the C,,, carotenoids, and a more complicated reaction mechanism is required than had been previously proposed [ 1J. The conformation of a molecule is usually more readily determined from the ‘H than from the “C spectrum but care has to be exercised to establish the precise identity of groups that give rise to particular “C and ‘H signals. For example, the homologous geminal Me signals of the phytohormone abscisic acid reverse their relative chemical shifts between ‘H and 13C spectra [7].
Chemical shifts obtained in [*H,J benzene are given in Table 1. The signal observed to be enriched by incorporation of [2-“C] mevalonate was that at 22.54 ppm in [*HI chloroform (Englert, unpublished results cited by B&ton [SJ) and was identified as that at 22.71 ppm in [*Ha] benzene, its downfield partner being found at 28.4 ppm (chloroform) and 27.64 ppm (benzene). A 13C-*H correlation experiment showed that the relative positions of the geminal C-l methyl signals were the same in both spectra. The upfield signals in the proton spectrum (1.05 ppm) correlated with those in the “C spectrum (22.71 ppm), and likewise for the downfield signals (1.21 ppm in the ‘H spectrum, correlated with the signal at 27.64 ppm in the ‘“C spectrum). The proton signal from C-2 was identified at 1.42 ppm (45.8 ppm in t3C spectra). The protons at C-l” were found to give separate signals at 1.86 and 2.26 ppm, thereby indicating that there was very restricted rotation about the C-2-Cl” bond. Correlated 20 spectroscopy (COS Y)
Analysis of the COSY spectrum revealed geminul coup lings between the two C-l” protons (1.86 and 2.26 ppm). The proton at C-2 was coupled to only one of the protons at C-l” (1.86 ppm), and coupling was seen between the C2 and one of the C-3 protons (1.77 ppm). Coupling between the C-3 protons (1.77 and 1.41 ppm) and between the geminal C-3 protons was also observed, and between the C-3 protons (1.77 ppm and 1.41 ppm) and the protons at C-4 (2.00 ppm). In addition, coupling between the Me at C-5 (1.81 ppm) was observed to the C-4 signal. NOE experiments
A ‘H NOE by difference experiment gave several small enhancements when either of the geminal-Me-l signals were irradiated, in particular those at 6.35 ppm (protons at C-7, C-8), and the C-l” H signal at 2.26 ppm, but not that of the other C-l” H at 1.86 ppm. Also, when the upfield Me was irradiated, a very small enhancement of the signal at 1.42 ppm was observed; when the downfield Me was irradiated small enhancements were observed for the signals at 1.61 ppm (C5” methyl) and at 1.77 ppm (C-3). Irradiation at 1.42 ppm, however, gave a large enhancement of the methyl signal at 1.05 ppm, i.e. the
S. H. RHODES and B. V MILB~RROW
934 REFERENCES
1. Eugster, C. H. (1979) Pure Appl. Chem. 51,463. 2. Britton, G., Lockely, W. J. S., Patel, N. J., Goodwin, T. W. and Englert, G. (1977) J. Chem. Sot. Chem. Commun. 655. 3. Britton, G., Goodwin, T. W., Lockley, W. J. S., Mundy, A. P., Pate], N. J. and Englert, G. (1979) J. Chem. Sot. Chem. Commun. 27. 4. Englert, G. (1982) in Carotenoid Chemistry and Eiochemistry (Britton, G. and Goodwin, T. W., eds), p. 107. Pergamon Press, Oxford. 5. Britton, G. (1985) Pure Appl. Chem. 57, 701. 6. Britton, G. and Mundy, A. P. (1980) Dev. Plant Biol. 6, 345.
7. Milborrow, B. V. (1984) Biochem. J 220, 325. S. and Weeks, 0. B. 8. Andrewes, A. C., Liaaen-Jensen, (1975) Acta Chem. Scand. 29, 884. 9. Milborrow, B. V. (1983) in Biosynthesis ofIsoprenoid Compounds (Porter, J. W. and Spurgeon, S. L., eds), Vol. 2, pp. 413-436. John Wiley, New York. Starr, M. P. and Sapirstein, S. (1953) Arch. Biochem. lo. Biophys. 43, 157. S. (1970) 11. Norgard, S., Aasen, A. J. and Liaaen-Jenson, Acta Chem. Scand. 24, 2183. 12. Britton, G. (1986) Meths in Enzymol. 111, 113.