[4]
SERS SPECTROSCOPY
31
Harashima et al. ~° has been confirmed by MS, IR, and ~H NMR (Fig. 5) data. 1 Papilioerythrinone possesses two chiral centers at C-3 and C-6'. The absolute configuration was determined by CD spectral data (Fig. 6) and comparison of NaBH4 reduction products with those of fritschiellaxanthin [(3S, 3'R,6'R)-3,Y-dihydroxy-fl, e-caroten-4-one] 12,13 and a-doradexanthin [(3S,3'S,6'R)-3,3'-dihydroxy-fl, e-caroten-4-one]. 12 The CD spectrum of papilioerythrinone was closely similar to those of fritschiellaxanthin and a-doradexanthin (Fig. 6). Therefore it was assumed that papilioerythrinone possesses the same chiralities at C-3 and C-6' as those of fritschieUaxanthin and a-doradexanthin. Reduction of papilioerythrinone (0.6 mg) with NaBH4 (20 mg) in methanol (20 ml) for 10 min at 20 ° provided four stereoisomers of triols (1), (2), (3), and (4) on Fig. 5, which were separated by HPLC on Sumipax OA-2000 with n-hexane-CH2C12-ethanol (48 : 16: 1.5) as shown in Fig. 5. As was expected, triols (1) and (2) were completely identical to triols (1') and (2') derived from fritschiellaxanthin by NaBH4 reduction, respectively, and triols (3) and (4) also completely coincided with triols (3') and (4') from a-doradexanthin (Fig. 5). Thus the absolute configuration ofpapilioerythrinone was determined to be (3S,6'R)-configuration by the accumulated evidence described above. L0K. Harashima, J. Nakahara, and G. Kato, Agric. Biol. Chem. 40, 711 (1976). " G. Englert, in "Carotenoid Chemistry and Biochemistry" (G. Britton and T. W. Goodwin, eds.), Proc. 6th Int. Symp. Carotenoids, p. 107. Pergamon, Oxford, 1982. 12 R. Buchecker, C. H. Eugster, and A. Weber, Helv. Chim. Acta 61, 1962 (1978). ~3T. Matsuno and M. Ookubo, Nippon Suisan Gakkaishi 48, 653 (1982).
[4] S u r f a c e - E n h a n c e d of Photosynthetic
Raman Scattering Spectroscopy Membranes and Complexes
By M I C H A E L SEIBERT, R A F A E L PICOREL, J A E - H O K I M , and THERESE M . COTTON
Introduction Surface-enhanced Raman scattering (SERS) results when ion complexes, molecules, or chromophores adsorbed onto or near roughened silver, gold, or copper substrates are excited with laser light. The effect was first reported for pyridine on anodized silver electrodes ~-3 where an enM. Fleischmann, P. F. Hendra, and A. J. McQuillan, Chem. Phys. Lett. 26, 123 (1974).
METHODSIN ENZYMOLOGY,VOL. 213
Copyright© 1992by AcademicPress,Inc. Allrightsof reproductionin any formreserved.
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CHEMISTRY: SYNTHESIS, PROPERTIES, CHARACTERIZATION
[4]
hancement of 106-fold over normal Raman scattering intensity was determined. 2,3 Both chemical and electromagnetic interactions between the adsorbate and the metal contribute to the enhancement phenomenon, depending on the metal, the adsorbate, and the surface structure? Surfaceenhanced resonance Raman scattering (SERRS) occurs when the laser excitation wavelength is in resonance with an electronic transition in the adsorbate and provides additional enhancement. The major advantages of SERS/SERRS include extreme analytical sensitivity, molecular selectivity, minimization of fluorescence, and distance sensitivity. Thus, sample concentrations in the range that is used for fluorescence spectroscopy are appropriate, and chromophores can be selected for or discriminated against, depending on the excitation wavelength. Since the Raman intensity is proportional to (a/r), ~° where a is the local radius of curvature at the metal surface and r is the distance of the adsorbate from the center of the local radius of curvature, only scattering centers located within tens of angstroms are detectable? Interference by Rayleigh scattering generally is not a problem because Raman scattering is inelastic (scattering is shifted by vibrational frequencies), although SERS/SERRS is limited to the nearultraviolet (UV) through the near-infrared (IR) region of the spectrum. Although many small molecules have been studied by SERS/SERRS, the investigation of complex biomolecules has been limited. Still, spectra for amino acids, proteins, nucleic acids, polynucleotides, DNA, and lipid monolayers have been reported. 6-9 Valuable information about surface interactions, the types of chemical structures leading to enhancement, electron transfer, denaturation, redox effects, enzyme activity, selective chromophore excitation, spin states, distance effects, and frequency shifts has been obtained. 7-9 All of this work set the stage for the application of SERS/SERRS to membrane systems ~°,H and their components, which by virtue of their complexity are well suited to investigation by a sensitive, selective, distance-dependent technique. 2 D. L. Jeanmaire and R. P. Van Duyne, J. Electroanal. Chem. 84, 1 (1977). 3 M. G. Albrecht and J. A. Creighton, J. Am. Chem. Soc. 99, 5215 (1977). 4 H. Metiu, in "Surface Enhanced Raman Scattering" (R. K. Chang and T. E. Furtak, eds.), p. 1. Plenum, New York, 1982. T. M. Cotton, R. A. Uphaus, and D. M6bius, J. Phys. Chem. 90, 6071 (1986). 6 I. R. Nabiev, S. D. Trakhanov, E. S. Efremov, V. V. Marinyuk, and R. M. LasorenkoManevich, Bioorg. Khim. 7, 941 (1981). 7 T. M. Cotton, Adv. Spectrosc. 15, 91 (1988). s E. Koglin and J.-M. S&tuaris, Top. Curr. Chem. 134, 1 (1986). 9 I. R. Nabiev, R. G. Efremov, and G. D. Chumanov, Sov. Phys.-- Usp. (Engl. Transl.) 31, 241 (1988). to M. Seibert and T. M. Cotton, FEBSLett. 182, 34 (1985). ~ I. R. Nabiev, R. G. Efremov, and G. D. Chumanov, J. Biosci. 8, 363 (1985).
[4]
SERS SPECTROSCOPY
33
Experimental P r o c e d u r e s
Raman Instrumentation As in conventional Raman and resonance Raman (RR) spectroscopy, lasers are used to excite SERS/SERRS. In most instances, continuous-wave gas lasers, such as Ar + or Kr +, have been used, although pulsed lasers may be used as well. The advantage of the latter is that time-resolved spectra are obtained, and these can provide mechanistic information. The two most common sample illumination geometries for solution Raman and R R samples seen in Fig. 1A and B involve collection of the scattered light at 90 ° relative to the incident angle or in the backscattering mode. In SERS/ SERRS, the laser light is focused on the sample (adsorbed on a substrate, see below) at an incident angle of approximately 60 °, as seen in Fig. 1C. The scattered radiation is collected in the backscattering mode by a lens with a high collection efficiency. The light is then collimated through a second lens with a n f n u m b e r that matches the spectrometer. The choice of spectrometer and detector is particularly important when applying SERS/SERRS to biological molecules. This is especially true when electrodes are used as the SERS-active substrate. Irradiation of the sample can lead to rapid photodegradation of the biomolecule. Rotation of the electrode in the laser beam can help to alleviate this problem, but the use of multichannel detection schemes is another important approach. With array detectors or charge-coupled devices, a substantial part of the spectrum [e.g., 850 cm -~ with an 1200-g/mm grating (Triplemate 1877, Spex Industries, Edison, NJ) and 514.5-nm excitation] can be collected within seconds. Figure 2 illustrates a monochromator/spectrograph combination with an attached diode array detector. The monochromator stage functions as a filter to remove Rayleigh light and transmit a band of frequencies to the spectrograph stage. The latter disperses the light across the detector. A typical diode array contains 1024 pixels, each of which is 25 pm in width and 2.5 mm in height.
SERS Substrate Preparation The three most common SERS substrates include electrodes, vacuumdeposited island films, and silver colloids. In all cases, some type of surface roughness is necessary to achieve maximal enhancement. The particulate nature of the surface provides enhancement of the electromagnetic field near the metal surface according to the electromagnetic theory of enhancement. The methods for preparing these substrates are summarized here. Silver electrodes are prepared by sealing flattened silver wire or foil in a glass tube. An inert resin, such as Torr Seal (Varian, Palo Alto, CA) is used
34
CHEMISTRY: SYNTHESIS, PROPERTIES, CHARACTERIZATION
[4]
A
0L2 ~
L1
S
L2
C S
FIG. 1. Schematic for Raman illumination geometry. (A) 90 ° illumination: S is the sample (cuvette), Lt is the focusing lens for the laser beam, and L2 is the collection lens for the Raman scattered light. (B) 180 ° or backscattering illumination: S is the sample (test tube, cuvette, electrochemical cell, etc.), Lt is a focusing lens (often a cylindrical lens is used to produce a line image at the spectrometer slit), L2 is the collection lens, and M is a small front-surface mirror that directs the laser beam to the sample. (C) Same as (B), except the laser beam is directed into the sample at an angle of 60 °.
[4]
SERS SPECTROSCOPY
35
E H
I
A
I
B
I
C
FIG. 2. Diagram of Raman instrumentation. A, Argon ion laser; B, krypton ion laser; C, mirrors; D, tunable premonochromater (Anaspec, Applied Photophysics,Hudson, MA); E, sample holder; F, collectinglens; G, focusinglens; H, monochromator stage of spectrometer (Spex Triplemate); I, spectrograph stage of spectrometer (Spex Triplemate); J, photodiode array detector (PARC 1420, Princeton Applied ResearchCorp., Princeton, NJ); K, detector controller (PARC 1418);L, PARCOMA II operating systemand computer. for this purpose. The surface is then polished with an alumina suspension in water, beginning with a coarse grade (5/tm) and ending with the finest grade (0.05 gm). A polishing wheel equipped with a nylon disk may be used for this purpose. At this point, the surface is mirror-like in appearance. The electrode is roughened by an oxidation-reduction cycle (ORC) in an electrochemical cell. Figure 3 shows a typical spectroelectrochemical cell containing the silver working electrode, a counter electrode (usually platinum), and a reference electrode, such as the standard calomel electrode (SCE). The potentials used for the ORC depend on the electrolyte and the size of the electrode. When 0.1 M Na2SO4 is used, a typical cycle involves stepping the potential from - 0 . 6 to +0.45 V and allowing the silver to oxidize until approximately 25 mC/cm 2 of charge has passed. At this point, the electrode is stepped back to the starting potential, and the Ag+ in the diffusion layer is reduced on the electrode surface. This produces a roughened surface containing particles approximately 250 A in diameter. Silver island films are prepared by vacuum (< 1 × 10-6 torr) deposition of silver (utilizing resistive heating) onto a surface such as glass or quartz
36
CHEMISTRY: SYNTHESIS, PROPERTIES, CHARACTERIZATION A
G
[4]
A
0
E.
Front View
Side View
Fxo. 3. Diagram of SERRS electrochemical cell. A, SERRS electrode submerged in cell; B, thermometer damp; C, cell clamp assembly; D, cell body openings; E, nitrogen blanket inlet; F, platinum auxiliary electrode; G, front window of glass; H, O rings; I, rear plate; J, SERRS electrode surface.
slides. The rate of deposition, temperature of the glass substrate, and the thickness of the film are all important parameters with respect to the SERS activity of these surfaces. Typically, the films are prepared by depositing 50 A (average mass) of silver at a rate of 0.02-5 A/sec. Under these conditions, silver islands are formed with an average diameter of about 200-400 A. The silver film is dipped into a solution of the molecule of interest for a sufficiently long period of time to adsorb the sample. The dipping time and solution concentration determine the surface coverage. Silver colloids are prepared by reducing a solution of AgNO 3 with a reducing agent, such as sodium citrate or sodium borohydride. Although this is a relatively simple procedure, it is essential that the container be scrupulously clean. If not, the silver sol will aggregate and precipitate.
Photosynthetic Samples A number of different photosynthetic protein-pigment complexes and membrane preparations have been examined by SERRS. These include isolated reaction center (RC) complexes, 12.13chromatophores, and spheroplasts from photosynthetic bacteria and their mutants, ~4a5 and photosystern II preparations. ~°,~3 Electrodes were used in each case. Following the 12T. M. Cotton and R. P. Van Duyne, FEBSLett. 147, 81 (1982). 13 R. Picorel, R. E. Holt, R. Heald, T. M. Cotton, and M. Seibcrt, J. Am. Chem. Soc. 113,
2839 (1991). 14R. Picorel, R. E. Holt, T. M. Cotton, and M. Seibert, J. Biol. Chem. 263, 4374 (1988). 1~R. Picorel, T. Lu, R. E. Holt, T. M. Cotton, and M. Scibcrt, Biochemistry 29, 707 (1990).
[4]
SERS SPECTROSCOPY
37
roughening procedure, the samples were added to the electrochemical cell containing electrolyte buffer solution and the electrode, or the electrode was dipped into a solution containing sample. In the first case, spectra were obtained after about 15 min to allow for adsorption of the biological preparation onto the electrode surface. With the second procedure, the electrode was removed after a short period of time and then placed in the electrochemical cell containing electrolyte-buffer solution for spectral analysis. The potential of the electrode should be adjusted to the appropriate value for obtaining the spectrum of interest. In some cases, samples do not adhere well, and the electrode cannot be placed into an electrolyte solution without loss of sample from the surface. Low-temperature methods may then be used. In this case, the silver electrode can be dipped in sample solution, and excess liquid removed either by gently touching a tissue to the surface, allowing the liquid to wet the tissue by capillary action, or by shaking excess liquid off the electrode surface. The electrode can then be submerged in a Dewar flask containing liquid nitrogen for spectral analysis.
Methods for Obtaining Data on Photosynthetic Samples The excitation wavelength and laser power are critical parameters for obtaining strong SERRS spectra. When samples contain multiple chromophores (e.g., chlorophylls a and b, carotenoids), it is possible to excite selectively only one class of molecules by choosing a wavelength that is in resonance with this class only. For example, in the case of photosynthetic bacterial preparations, excitation from 457.9 to 514.5 nm preferentially excites RR scattering from carotenoids. Laser power can have a dramatic effect if the sample is easily photodegraded. In all cases minimal power is used, and the integration time is adjusted to obtain good signal-to-noise ratios. Typically, the laser power is 20 mW or less. Other important variables include the electrode potential and temperature. Adsorption depends on the surface charge of the sample. In the case of certain samples, the adsorption interaction may change with potential if different regions of the surface have different charged groups. For example, in the case of bacterial RC preparations, adsorption appears to occur closest to the bacteriochlorophyll (BChl) molecules at negative potentials and closest to bacteriopheophytin molecules at more positive potentials. ~2 The temperature of the electrode can have a strong influence on the stability of photolabile samples. For some samples, it was determined that direct immersion of the electrode into liquid nitrogen produced the most stable spectra and preserved the native structure of the proteins. 16 16 T. M. Cotton, V. Sclflegel, R. E. Holt, B. Swanson, and P. Ortiz de Montellano, Proc. Int. Soc. Opt. Eng. 1055, 263 (1989).
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CHEMISTRY: SYNTHESIS, PROPERTIES, CHARACTERIZATION
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The presence of extraneous chemicals (buffers, electrolyte) can also play a role in SERRS of photosynthetic samples. Buffers, such as Tris, can produce strong scattering and interference bands. Counterions, such as chloride, are important with respect to sample adsorption and the production of SERRS-active sites) 7 In most cases, the samples and the electrolyte solution are degassed to minimize reactions with oxygen. Potential problems that may occur in SERRS studies include lack of adsorption of the sample to the metal surface, photoinstability (bleaching or photoreactivity), and disruption of the protein structure on the electrode surface with time. Adsorption can be affected by the potential of the metal surface. This can be varied by a potentiostat (electrode) or by adding extraneous small molecules that adsorb to the surface and change the surface potential (e.g., chloride ions or citrate on silver sols). Again, photoinstability and structural changes can be minimized by using lowtemperature techniques. The use of a multichannel detector is also important because collection time can be drastically reduced in comparison to that required for single-channel detection. Finally, it is necessary that the samples and water used for the spectral analysis be as free from contaminants as possible. Often, impurities can produce strong SERS/SERRS spectra and/or prevent adsorption of the sample to the surface. Examples of S E R R S Results Obtained for Carotenoids
Bacterial Photosynthetic Systems Location of Carotenoids in Chromatophore Membranes. Photosynthetic membranes isolated from Rhodospirillum (Rs.) rubrum have been examined by SERRS in liquid electrolyte suspensions. Chromatophore (cytoplasmic side out) and spheroplast (periplasmic side out) vesicles from wild-type (S 1), carotenoidless mutant (G9), and reaction centerless mutant (F24) strains were used to locate spirilloxanthin (Spx) on the cytoplasmic side of the membrane within the B880 antenna complex) 4 The extreme distance-sensitive property of SERRS allowed observation of Spx peaks at 1508, 1151, and 1001 cm -~ with chromatophores but not spheroplasts. This was instrumental in drawing a modeP 4 for B880 in which the carotenoid partially spans the membrane, exposed on the cytoplasmic side but embedded near BChl molecules at some distance from the periplasmic side. Similar observations have been made with the carotenoids in the antenna complexes of Rhodobacter (Rb.) sphaeroides membranes.t5 17 p. Hildebrandt and M. Stockburger, or. Phys. Chem. 88, 5935 (1984).
[4]
SERS SPECTROSCOPY
39
Orientation of Carotenoids in Chromatophore Membranes. In certain cases it is also possible to ascertain orientational information on asymmetric carotenoid molecules by comparing SERRS and R R frequency peaks? 5 This was demonstrated in liquid suspensions by comparing chromatophores isolated from photosynthetically grown Rb. sphaeroides and those grown aerobically in the dark. Spheroidene with a methoxy group on one end is synthesized in the former case, and spheroidenone with an oxo group on the methoxy end is synthesized in the latter. SERRS and R R peaks observed for spheroidene were the same (1519, 1153, and 1000 cm-~), whereas the 1511 cm -l R R peak observed for spheriodenone shifted to 1520 cm -~ in the case of SERRS (Fig. 4). The shift most likely results from interaction of the oxo group with the silver electrode in a manner that disrupts its conjugation with the polyene chain of the carotenoid and thus restricts delocalization of the 7t electrons. This demonstrates that the methoxy end of the carotenoids is oriented toward the cytoplasmic side of the chromatophore membrane, because the periplasmic side gives no SERRS signal. ~5 Reaction Center and Antenna Complexes. Low-temperature SERRS techniques have been used to examine spirilloxanthin in isolated RC and antenna complexes. Peaks at 1526, 1156, and 1000 cm-~ in RC complexes are characteristic of the cis isomer, whereas peaks at 1506- 1510, 1149, and 1001 cm -1 in chromatophores and acetone-extracted spirilloxanthin are indicative of the all-trans conformation? 3 Carotenoid spectra in isolated antenna complexes are quite similar to those obtained with chromatophores (Picorel, Cotton, and Seibert, unpublished results). Large carotenoid signals obtained in RCs indicate that the molecule is located close to the surface, in agreement with X-ray crystallographic data.18 It is noteworthy that the detergents [lauryl dimethylamine N-oxide (LDAO), Triton X-100, and sodium dodecyl sulfate (SDS)] used to isolate the pigmentprotein complexes have little effect on the SERRS signals. Green Plant Photosynthetic Systems Location of Carotenoids in Thylakoid Membranes. Examination of stromal- and lumenal-side-out spinach thylakoid vesicles and (stromal side out) photosystem II (PSII) membrane fragments using liquid electrolyte SERRS techniques led to the conclusion that carotenoids are exposed on both sides of higher plant membranes? 9 Binding of antibodies (raised ~sT. O. Yeates, H. Komiya, A. Chirino, D. C. Rees, J. P. Allen, and G. Feher, Proc. Natl. Acad. Sci. U.S.A.85, 7993 (1988). 19M. Bakhtiari, R. Picorel, T. M. Cotton, and M. Seibert, Photochem. Photobiol., in press (1992).
40
CHEMISTRY: SYNTHESIS, PROPERTIES, CHARACTERIZATION
[4]
A
1512 A 45.._.,7nnm/ .9 1511
1150 1151 _
~
~
1003
¢,. w
1512 514.5 A nmj
K
_
.
1151 ~
530.9nm 1510
IB
1520 1520
1149
1157
100(
1
1156
E 1518 514.5 , ,
1155 A
~ 1
Raman Shift (cm-1)
Fla. 4. Resonance Raman (A) and SERRS (B) spectra of chromatophores isolated from wild-typeRb. sphaeroides grown aerobicallyin the dark. Note the shift in peak frexluencyin the SERRS spectra, indicating that the methoxyl end of spheroidenone is located on the
cytoplasmicside of the membrane.
against lumenal-side proteins) to PSII membrane fragments confirmed the distance sensitivity of SERRS and led to the conclusion that carotenoids are also exposed at the appressed membrane surfaces of thylakoids. 19 Isolated Reaction Centers. Low-temperature SERRS studies revealed that fl-carotene, the only carotenoid present in isolated D 1/D2 PSII RC,
[4]
SERS SPECTROSCOPY
41
was located near the surface of the complex and was in the all-trans configuration (peaks at 1527, 1158, and 1006 cm-~), consistent with RR data. Again, the presence of detergents did not prevent detection of SERRS signals. Do S E R R S Techniques D e n a t u r e Protein Complexes? To observe SERRS signals, protein complexes must be adsorbed onto anodized metal surfaces. There has been a long-standing debate over whether physical and/or chemical interactions between the two disrupt protein structure (and hence affect conclusions obtained from biological SERRS studies). The fact that the cis conformation of the native bacterial RC carotenoid is maintained during SERRS experiments with isolated RCs and no new peaks are ascribed to heme denaturation of cytochrome b-559 in isolated PSII RC complex suggested that significant protein denaturation does not occur under the conditions used. ~a Although this is encouraging, a general rule cannot as yet be drawn, and every protein complex should be considered individually. Other Applications of S E R S / S E R R S in Photosynthesis Although emphasis in this chapter has been on carotenoids, similar SERRS techniques can be used to examine other photosynthetic chromophores. Cytochrome b-559 has been mentioned above, but other pigments, including other cytochromes, chlorophylls and other porphyrins, flavins, antenna pigments in addition to chlorophylls and carotenoids, quinones, and other electron-transport components, are under study. ~3,2° SERS signals attributable indirectly to the presence of manganese, functional in the O2-evolution process of photosynthesis, have also been observed. ~°,2~ Finally, SERS spectra of bacteriorhodopsin in purple membrane have been recorded. 9 Conclusions and F u t u r e Developments The results described above demonstrate that SERRS has considerable potential for analytical applications (determination of Raman spectra on very small and highly dilute samples), as well as for fundamental studies of 20 R. Picorel, T. Lu, R. E. Holt, T. M. Cotton, and M. Seibert, in "Current Research in Photosynthesis" (M. Baltscheffsky, ed.), Vol. II, p. 907. Kluwer Academic, Dordrecht, The Netherlands, 1990. 21 M. Seibert, T. M. Cotton, and J. G. Metz, Biochim. Biophys. Acta 934, 235 0988).
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CHEMISTRY: SYNTHESIS, PROPERTIES, CHARACTERIZATION
[5]
biomolecular structure on surfaces. The membrane studies suggest that SERRS can provide qualitative information about the location and orientation of chromophores within the membrane. Future developments will include the use of new detectors (e.g., charge-coupled devices), which have very low dark counts and can be used to integrate signals for long time periods. It should be possible to obtain unenhanced Raman spectra on any surface using this approach. Although no longer surface (distance) sensitive, this approach will provide complementary information to SERRS about the structure of macromolecules on surfaces. Also, the use of new lasers, such as the titanium: sapphire laser, will allow excitation throughout the red region of the electromagnetic spectrum (650-1000 nm). This will be valuable for studies of biological samples on gold substrates. Finally, new nonlinear techniques, such as hyper-Raman and surface-enhanced hyper-Raman scattering, will be possible using pulsed excitation. Acknowledgment This work was supported by the Divisions of Chemical Sciences (M.S. and T.M.C.) and Energy Biosciences (M.S.), Office of Basic Energy Sciences, U.S. Department of Energy; Consejo Superior de Investigaciones Cientificas, Spain (R.P.); and the National Institutes of Health (T.M.C., grant Number GM-35108). Ames Laboratory is operated for the U.S. Department of Energy by Iowa State University under Contract No. W-7405-Eng-82. SERI is a division of the Midwest Research Institute and operated for the U.S. Department of Energy under Contract No. DE-AC02-83CH-10093.
[5] S y n t h e s i s o f C a r o t e n o i d s S p e c i f i c a l l y L a b e l e d w i t h Isotopic Carbon and Tritium
By ARNOLD A. LIEBMAN, WALTER BURGER,
SATISH C. CHOUDHRY,
and
JOSEPH CUPANO
Introduction The chemistry used in the synthesis of carotenoids is quite extensive and has been periodically reviewed, l This literature reflects the expanding use of chiral reagents and other tools of modern organic synthesis and also the increasing use of isotopically labeled carotenoids in a variety of applit K. Bernhard, in "Carotenoids: Chemistry and Biology" (N. I. Krinsky, M. M. MathewsRoth, and R. F. Taylor, eds.), Proc. 8th Int. Syrup. Carotenoids, p. 337. Plenum, New York, 1989. METHODSIN ENZYMOLOGY,VOL.213
Copyright© 1992byAcademicPress,Inc. Allrightsofreproductionin anyformreserved.
SERS SPECTROSCOPY
31
Harashima et al. ~° has been confirmed by MS, IR, and ~H NMR (Fig. 5) data. 1 Papilioerythrinone possesses two chiral centers at C-3 and C-6'. The absolute configuration was determined by CD spectral data (Fig. 6) and comparison of NaBH4 reduction products with those of fritschiellaxanthin [(3S, 3'R,6'R)-3,Y-dihydroxy-fl, e-caroten-4-one] 12,13 and a-doradexanthin [(3S,3'S,6'R)-3,3'-dihydroxy-fl, e-caroten-4-one]. 12 The CD spectrum of papilioerythrinone was closely similar to those of fritschiellaxanthin and a-doradexanthin (Fig. 6). Therefore it was assumed that papilioerythrinone possesses the same chiralities at C-3 and C-6' as those of fritschieUaxanthin and a-doradexanthin. Reduction of papilioerythrinone (0.6 mg) with NaBH4 (20 mg) in methanol (20 ml) for 10 min at 20 ° provided four stereoisomers of triols (1), (2), (3), and (4) on Fig. 5, which were separated by HPLC on Sumipax OA-2000 with n-hexane-CH2C12-ethanol (48 : 16: 1.5) as shown in Fig. 5. As was expected, triols (1) and (2) were completely identical to triols (1') and (2') derived from fritschiellaxanthin by NaBH4 reduction, respectively, and triols (3) and (4) also completely coincided with triols (3') and (4') from a-doradexanthin (Fig. 5). Thus the absolute configuration ofpapilioerythrinone was determined to be (3S,6'R)-configuration by the accumulated evidence described above. L0K. Harashima, J. Nakahara, and G. Kato, Agric. Biol. Chem. 40, 711 (1976). " G. Englert, in "Carotenoid Chemistry and Biochemistry" (G. Britton and T. W. Goodwin, eds.), Proc. 6th Int. Symp. Carotenoids, p. 107. Pergamon, Oxford, 1982. 12 R. Buchecker, C. H. Eugster, and A. Weber, Helv. Chim. Acta 61, 1962 (1978). ~3T. Matsuno and M. Ookubo, Nippon Suisan Gakkaishi 48, 653 (1982).
[4] S u r f a c e - E n h a n c e d of Photosynthetic
Raman Scattering Spectroscopy Membranes and Complexes
By M I C H A E L SEIBERT, R A F A E L PICOREL, J A E - H O K I M , and THERESE M . COTTON
Introduction Surface-enhanced Raman scattering (SERS) results when ion complexes, molecules, or chromophores adsorbed onto or near roughened silver, gold, or copper substrates are excited with laser light. The effect was first reported for pyridine on anodized silver electrodes ~-3 where an enM. Fleischmann, P. F. Hendra, and A. J. McQuillan, Chem. Phys. Lett. 26, 123 (1974).
METHODSIN ENZYMOLOGY,VOL. 213
Copyright© 1992by AcademicPress,Inc. Allrightsof reproductionin any formreserved.
32
CHEMISTRY: SYNTHESIS, PROPERTIES, CHARACTERIZATION
[4]
hancement of 106-fold over normal Raman scattering intensity was determined. 2,3 Both chemical and electromagnetic interactions between the adsorbate and the metal contribute to the enhancement phenomenon, depending on the metal, the adsorbate, and the surface structure? Surfaceenhanced resonance Raman scattering (SERRS) occurs when the laser excitation wavelength is in resonance with an electronic transition in the adsorbate and provides additional enhancement. The major advantages of SERS/SERRS include extreme analytical sensitivity, molecular selectivity, minimization of fluorescence, and distance sensitivity. Thus, sample concentrations in the range that is used for fluorescence spectroscopy are appropriate, and chromophores can be selected for or discriminated against, depending on the excitation wavelength. Since the Raman intensity is proportional to (a/r), ~° where a is the local radius of curvature at the metal surface and r is the distance of the adsorbate from the center of the local radius of curvature, only scattering centers located within tens of angstroms are detectable? Interference by Rayleigh scattering generally is not a problem because Raman scattering is inelastic (scattering is shifted by vibrational frequencies), although SERS/SERRS is limited to the nearultraviolet (UV) through the near-infrared (IR) region of the spectrum. Although many small molecules have been studied by SERS/SERRS, the investigation of complex biomolecules has been limited. Still, spectra for amino acids, proteins, nucleic acids, polynucleotides, DNA, and lipid monolayers have been reported. 6-9 Valuable information about surface interactions, the types of chemical structures leading to enhancement, electron transfer, denaturation, redox effects, enzyme activity, selective chromophore excitation, spin states, distance effects, and frequency shifts has been obtained. 7-9 All of this work set the stage for the application of SERS/SERRS to membrane systems ~°,H and their components, which by virtue of their complexity are well suited to investigation by a sensitive, selective, distance-dependent technique. 2 D. L. Jeanmaire and R. P. Van Duyne, J. Electroanal. Chem. 84, 1 (1977). 3 M. G. Albrecht and J. A. Creighton, J. Am. Chem. Soc. 99, 5215 (1977). 4 H. Metiu, in "Surface Enhanced Raman Scattering" (R. K. Chang and T. E. Furtak, eds.), p. 1. Plenum, New York, 1982. T. M. Cotton, R. A. Uphaus, and D. M6bius, J. Phys. Chem. 90, 6071 (1986). 6 I. R. Nabiev, S. D. Trakhanov, E. S. Efremov, V. V. Marinyuk, and R. M. LasorenkoManevich, Bioorg. Khim. 7, 941 (1981). 7 T. M. Cotton, Adv. Spectrosc. 15, 91 (1988). s E. Koglin and J.-M. S&tuaris, Top. Curr. Chem. 134, 1 (1986). 9 I. R. Nabiev, R. G. Efremov, and G. D. Chumanov, Sov. Phys.-- Usp. (Engl. Transl.) 31, 241 (1988). to M. Seibert and T. M. Cotton, FEBSLett. 182, 34 (1985). ~ I. R. Nabiev, R. G. Efremov, and G. D. Chumanov, J. Biosci. 8, 363 (1985).
[4]
SERS SPECTROSCOPY
33
Experimental P r o c e d u r e s
Raman Instrumentation As in conventional Raman and resonance Raman (RR) spectroscopy, lasers are used to excite SERS/SERRS. In most instances, continuous-wave gas lasers, such as Ar + or Kr +, have been used, although pulsed lasers may be used as well. The advantage of the latter is that time-resolved spectra are obtained, and these can provide mechanistic information. The two most common sample illumination geometries for solution Raman and R R samples seen in Fig. 1A and B involve collection of the scattered light at 90 ° relative to the incident angle or in the backscattering mode. In SERS/ SERRS, the laser light is focused on the sample (adsorbed on a substrate, see below) at an incident angle of approximately 60 °, as seen in Fig. 1C. The scattered radiation is collected in the backscattering mode by a lens with a high collection efficiency. The light is then collimated through a second lens with a n f n u m b e r that matches the spectrometer. The choice of spectrometer and detector is particularly important when applying SERS/SERRS to biological molecules. This is especially true when electrodes are used as the SERS-active substrate. Irradiation of the sample can lead to rapid photodegradation of the biomolecule. Rotation of the electrode in the laser beam can help to alleviate this problem, but the use of multichannel detection schemes is another important approach. With array detectors or charge-coupled devices, a substantial part of the spectrum [e.g., 850 cm -~ with an 1200-g/mm grating (Triplemate 1877, Spex Industries, Edison, NJ) and 514.5-nm excitation] can be collected within seconds. Figure 2 illustrates a monochromator/spectrograph combination with an attached diode array detector. The monochromator stage functions as a filter to remove Rayleigh light and transmit a band of frequencies to the spectrograph stage. The latter disperses the light across the detector. A typical diode array contains 1024 pixels, each of which is 25 pm in width and 2.5 mm in height.
SERS Substrate Preparation The three most common SERS substrates include electrodes, vacuumdeposited island films, and silver colloids. In all cases, some type of surface roughness is necessary to achieve maximal enhancement. The particulate nature of the surface provides enhancement of the electromagnetic field near the metal surface according to the electromagnetic theory of enhancement. The methods for preparing these substrates are summarized here. Silver electrodes are prepared by sealing flattened silver wire or foil in a glass tube. An inert resin, such as Torr Seal (Varian, Palo Alto, CA) is used
34
CHEMISTRY: SYNTHESIS, PROPERTIES, CHARACTERIZATION
[4]
A
0L2 ~
L1
S
L2
C S
FIG. 1. Schematic for Raman illumination geometry. (A) 90 ° illumination: S is the sample (cuvette), Lt is the focusing lens for the laser beam, and L2 is the collection lens for the Raman scattered light. (B) 180 ° or backscattering illumination: S is the sample (test tube, cuvette, electrochemical cell, etc.), Lt is a focusing lens (often a cylindrical lens is used to produce a line image at the spectrometer slit), L2 is the collection lens, and M is a small front-surface mirror that directs the laser beam to the sample. (C) Same as (B), except the laser beam is directed into the sample at an angle of 60 °.
[4]
SERS SPECTROSCOPY
35
E H
I
A
I
B
I
C
FIG. 2. Diagram of Raman instrumentation. A, Argon ion laser; B, krypton ion laser; C, mirrors; D, tunable premonochromater (Anaspec, Applied Photophysics,Hudson, MA); E, sample holder; F, collectinglens; G, focusinglens; H, monochromator stage of spectrometer (Spex Triplemate); I, spectrograph stage of spectrometer (Spex Triplemate); J, photodiode array detector (PARC 1420, Princeton Applied ResearchCorp., Princeton, NJ); K, detector controller (PARC 1418);L, PARCOMA II operating systemand computer. for this purpose. The surface is then polished with an alumina suspension in water, beginning with a coarse grade (5/tm) and ending with the finest grade (0.05 gm). A polishing wheel equipped with a nylon disk may be used for this purpose. At this point, the surface is mirror-like in appearance. The electrode is roughened by an oxidation-reduction cycle (ORC) in an electrochemical cell. Figure 3 shows a typical spectroelectrochemical cell containing the silver working electrode, a counter electrode (usually platinum), and a reference electrode, such as the standard calomel electrode (SCE). The potentials used for the ORC depend on the electrolyte and the size of the electrode. When 0.1 M Na2SO4 is used, a typical cycle involves stepping the potential from - 0 . 6 to +0.45 V and allowing the silver to oxidize until approximately 25 mC/cm 2 of charge has passed. At this point, the electrode is stepped back to the starting potential, and the Ag+ in the diffusion layer is reduced on the electrode surface. This produces a roughened surface containing particles approximately 250 A in diameter. Silver island films are prepared by vacuum (< 1 × 10-6 torr) deposition of silver (utilizing resistive heating) onto a surface such as glass or quartz
36
CHEMISTRY: SYNTHESIS, PROPERTIES, CHARACTERIZATION A
G
[4]
A
0
E.
Front View
Side View
Fxo. 3. Diagram of SERRS electrochemical cell. A, SERRS electrode submerged in cell; B, thermometer damp; C, cell clamp assembly; D, cell body openings; E, nitrogen blanket inlet; F, platinum auxiliary electrode; G, front window of glass; H, O rings; I, rear plate; J, SERRS electrode surface.
slides. The rate of deposition, temperature of the glass substrate, and the thickness of the film are all important parameters with respect to the SERS activity of these surfaces. Typically, the films are prepared by depositing 50 A (average mass) of silver at a rate of 0.02-5 A/sec. Under these conditions, silver islands are formed with an average diameter of about 200-400 A. The silver film is dipped into a solution of the molecule of interest for a sufficiently long period of time to adsorb the sample. The dipping time and solution concentration determine the surface coverage. Silver colloids are prepared by reducing a solution of AgNO 3 with a reducing agent, such as sodium citrate or sodium borohydride. Although this is a relatively simple procedure, it is essential that the container be scrupulously clean. If not, the silver sol will aggregate and precipitate.
Photosynthetic Samples A number of different photosynthetic protein-pigment complexes and membrane preparations have been examined by SERRS. These include isolated reaction center (RC) complexes, 12.13chromatophores, and spheroplasts from photosynthetic bacteria and their mutants, ~4a5 and photosystern II preparations. ~°,~3 Electrodes were used in each case. Following the 12T. M. Cotton and R. P. Van Duyne, FEBSLett. 147, 81 (1982). 13 R. Picorel, R. E. Holt, R. Heald, T. M. Cotton, and M. Seibcrt, J. Am. Chem. Soc. 113,
2839 (1991). 14R. Picorel, R. E. Holt, T. M. Cotton, and M. Seibert, J. Biol. Chem. 263, 4374 (1988). 1~R. Picorel, T. Lu, R. E. Holt, T. M. Cotton, and M. Scibcrt, Biochemistry 29, 707 (1990).
[4]
SERS SPECTROSCOPY
37
roughening procedure, the samples were added to the electrochemical cell containing electrolyte buffer solution and the electrode, or the electrode was dipped into a solution containing sample. In the first case, spectra were obtained after about 15 min to allow for adsorption of the biological preparation onto the electrode surface. With the second procedure, the electrode was removed after a short period of time and then placed in the electrochemical cell containing electrolyte-buffer solution for spectral analysis. The potential of the electrode should be adjusted to the appropriate value for obtaining the spectrum of interest. In some cases, samples do not adhere well, and the electrode cannot be placed into an electrolyte solution without loss of sample from the surface. Low-temperature methods may then be used. In this case, the silver electrode can be dipped in sample solution, and excess liquid removed either by gently touching a tissue to the surface, allowing the liquid to wet the tissue by capillary action, or by shaking excess liquid off the electrode surface. The electrode can then be submerged in a Dewar flask containing liquid nitrogen for spectral analysis.
Methods for Obtaining Data on Photosynthetic Samples The excitation wavelength and laser power are critical parameters for obtaining strong SERRS spectra. When samples contain multiple chromophores (e.g., chlorophylls a and b, carotenoids), it is possible to excite selectively only one class of molecules by choosing a wavelength that is in resonance with this class only. For example, in the case of photosynthetic bacterial preparations, excitation from 457.9 to 514.5 nm preferentially excites RR scattering from carotenoids. Laser power can have a dramatic effect if the sample is easily photodegraded. In all cases minimal power is used, and the integration time is adjusted to obtain good signal-to-noise ratios. Typically, the laser power is 20 mW or less. Other important variables include the electrode potential and temperature. Adsorption depends on the surface charge of the sample. In the case of certain samples, the adsorption interaction may change with potential if different regions of the surface have different charged groups. For example, in the case of bacterial RC preparations, adsorption appears to occur closest to the bacteriochlorophyll (BChl) molecules at negative potentials and closest to bacteriopheophytin molecules at more positive potentials. ~2 The temperature of the electrode can have a strong influence on the stability of photolabile samples. For some samples, it was determined that direct immersion of the electrode into liquid nitrogen produced the most stable spectra and preserved the native structure of the proteins. 16 16 T. M. Cotton, V. Sclflegel, R. E. Holt, B. Swanson, and P. Ortiz de Montellano, Proc. Int. Soc. Opt. Eng. 1055, 263 (1989).
38
CHEMISTRY: SYNTHESIS, PROPERTIES, CHARACTERIZATION
[4]
The presence of extraneous chemicals (buffers, electrolyte) can also play a role in SERRS of photosynthetic samples. Buffers, such as Tris, can produce strong scattering and interference bands. Counterions, such as chloride, are important with respect to sample adsorption and the production of SERRS-active sites) 7 In most cases, the samples and the electrolyte solution are degassed to minimize reactions with oxygen. Potential problems that may occur in SERRS studies include lack of adsorption of the sample to the metal surface, photoinstability (bleaching or photoreactivity), and disruption of the protein structure on the electrode surface with time. Adsorption can be affected by the potential of the metal surface. This can be varied by a potentiostat (electrode) or by adding extraneous small molecules that adsorb to the surface and change the surface potential (e.g., chloride ions or citrate on silver sols). Again, photoinstability and structural changes can be minimized by using lowtemperature techniques. The use of a multichannel detector is also important because collection time can be drastically reduced in comparison to that required for single-channel detection. Finally, it is necessary that the samples and water used for the spectral analysis be as free from contaminants as possible. Often, impurities can produce strong SERS/SERRS spectra and/or prevent adsorption of the sample to the surface. Examples of S E R R S Results Obtained for Carotenoids
Bacterial Photosynthetic Systems Location of Carotenoids in Chromatophore Membranes. Photosynthetic membranes isolated from Rhodospirillum (Rs.) rubrum have been examined by SERRS in liquid electrolyte suspensions. Chromatophore (cytoplasmic side out) and spheroplast (periplasmic side out) vesicles from wild-type (S 1), carotenoidless mutant (G9), and reaction centerless mutant (F24) strains were used to locate spirilloxanthin (Spx) on the cytoplasmic side of the membrane within the B880 antenna complex) 4 The extreme distance-sensitive property of SERRS allowed observation of Spx peaks at 1508, 1151, and 1001 cm -~ with chromatophores but not spheroplasts. This was instrumental in drawing a modeP 4 for B880 in which the carotenoid partially spans the membrane, exposed on the cytoplasmic side but embedded near BChl molecules at some distance from the periplasmic side. Similar observations have been made with the carotenoids in the antenna complexes of Rhodobacter (Rb.) sphaeroides membranes.t5 17 p. Hildebrandt and M. Stockburger, or. Phys. Chem. 88, 5935 (1984).
[4]
SERS SPECTROSCOPY
39
Orientation of Carotenoids in Chromatophore Membranes. In certain cases it is also possible to ascertain orientational information on asymmetric carotenoid molecules by comparing SERRS and R R frequency peaks? 5 This was demonstrated in liquid suspensions by comparing chromatophores isolated from photosynthetically grown Rb. sphaeroides and those grown aerobically in the dark. Spheroidene with a methoxy group on one end is synthesized in the former case, and spheroidenone with an oxo group on the methoxy end is synthesized in the latter. SERRS and R R peaks observed for spheroidene were the same (1519, 1153, and 1000 cm-~), whereas the 1511 cm -l R R peak observed for spheriodenone shifted to 1520 cm -~ in the case of SERRS (Fig. 4). The shift most likely results from interaction of the oxo group with the silver electrode in a manner that disrupts its conjugation with the polyene chain of the carotenoid and thus restricts delocalization of the 7t electrons. This demonstrates that the methoxy end of the carotenoids is oriented toward the cytoplasmic side of the chromatophore membrane, because the periplasmic side gives no SERRS signal. ~5 Reaction Center and Antenna Complexes. Low-temperature SERRS techniques have been used to examine spirilloxanthin in isolated RC and antenna complexes. Peaks at 1526, 1156, and 1000 cm-~ in RC complexes are characteristic of the cis isomer, whereas peaks at 1506- 1510, 1149, and 1001 cm -1 in chromatophores and acetone-extracted spirilloxanthin are indicative of the all-trans conformation? 3 Carotenoid spectra in isolated antenna complexes are quite similar to those obtained with chromatophores (Picorel, Cotton, and Seibert, unpublished results). Large carotenoid signals obtained in RCs indicate that the molecule is located close to the surface, in agreement with X-ray crystallographic data.18 It is noteworthy that the detergents [lauryl dimethylamine N-oxide (LDAO), Triton X-100, and sodium dodecyl sulfate (SDS)] used to isolate the pigmentprotein complexes have little effect on the SERRS signals. Green Plant Photosynthetic Systems Location of Carotenoids in Thylakoid Membranes. Examination of stromal- and lumenal-side-out spinach thylakoid vesicles and (stromal side out) photosystem II (PSII) membrane fragments using liquid electrolyte SERRS techniques led to the conclusion that carotenoids are exposed on both sides of higher plant membranes? 9 Binding of antibodies (raised ~sT. O. Yeates, H. Komiya, A. Chirino, D. C. Rees, J. P. Allen, and G. Feher, Proc. Natl. Acad. Sci. U.S.A.85, 7993 (1988). 19M. Bakhtiari, R. Picorel, T. M. Cotton, and M. Seibert, Photochem. Photobiol., in press (1992).
40
CHEMISTRY: SYNTHESIS, PROPERTIES, CHARACTERIZATION
[4]
A
1512 A 45.._.,7nnm/ .9 1511
1150 1151 _
~
~
1003
¢,. w
1512 514.5 A nmj
K
_
.
1151 ~
530.9nm 1510
IB
1520 1520
1149
1157
100(
1
1156
E 1518 514.5 , ,
1155 A
~ 1
Raman Shift (cm-1)
Fla. 4. Resonance Raman (A) and SERRS (B) spectra of chromatophores isolated from wild-typeRb. sphaeroides grown aerobicallyin the dark. Note the shift in peak frexluencyin the SERRS spectra, indicating that the methoxyl end of spheroidenone is located on the
cytoplasmicside of the membrane.
against lumenal-side proteins) to PSII membrane fragments confirmed the distance sensitivity of SERRS and led to the conclusion that carotenoids are also exposed at the appressed membrane surfaces of thylakoids. 19 Isolated Reaction Centers. Low-temperature SERRS studies revealed that fl-carotene, the only carotenoid present in isolated D 1/D2 PSII RC,
[4]
SERS SPECTROSCOPY
41
was located near the surface of the complex and was in the all-trans configuration (peaks at 1527, 1158, and 1006 cm-~), consistent with RR data. Again, the presence of detergents did not prevent detection of SERRS signals. Do S E R R S Techniques D e n a t u r e Protein Complexes? To observe SERRS signals, protein complexes must be adsorbed onto anodized metal surfaces. There has been a long-standing debate over whether physical and/or chemical interactions between the two disrupt protein structure (and hence affect conclusions obtained from biological SERRS studies). The fact that the cis conformation of the native bacterial RC carotenoid is maintained during SERRS experiments with isolated RCs and no new peaks are ascribed to heme denaturation of cytochrome b-559 in isolated PSII RC complex suggested that significant protein denaturation does not occur under the conditions used. ~a Although this is encouraging, a general rule cannot as yet be drawn, and every protein complex should be considered individually. Other Applications of S E R S / S E R R S in Photosynthesis Although emphasis in this chapter has been on carotenoids, similar SERRS techniques can be used to examine other photosynthetic chromophores. Cytochrome b-559 has been mentioned above, but other pigments, including other cytochromes, chlorophylls and other porphyrins, flavins, antenna pigments in addition to chlorophylls and carotenoids, quinones, and other electron-transport components, are under study. ~3,2° SERS signals attributable indirectly to the presence of manganese, functional in the O2-evolution process of photosynthesis, have also been observed. ~°,2~ Finally, SERS spectra of bacteriorhodopsin in purple membrane have been recorded. 9 Conclusions and F u t u r e Developments The results described above demonstrate that SERRS has considerable potential for analytical applications (determination of Raman spectra on very small and highly dilute samples), as well as for fundamental studies of 20 R. Picorel, T. Lu, R. E. Holt, T. M. Cotton, and M. Seibert, in "Current Research in Photosynthesis" (M. Baltscheffsky, ed.), Vol. II, p. 907. Kluwer Academic, Dordrecht, The Netherlands, 1990. 21 M. Seibert, T. M. Cotton, and J. G. Metz, Biochim. Biophys. Acta 934, 235 0988).
42
CHEMISTRY: SYNTHESIS, PROPERTIES, CHARACTERIZATION
[5]
biomolecular structure on surfaces. The membrane studies suggest that SERRS can provide qualitative information about the location and orientation of chromophores within the membrane. Future developments will include the use of new detectors (e.g., charge-coupled devices), which have very low dark counts and can be used to integrate signals for long time periods. It should be possible to obtain unenhanced Raman spectra on any surface using this approach. Although no longer surface (distance) sensitive, this approach will provide complementary information to SERRS about the structure of macromolecules on surfaces. Also, the use of new lasers, such as the titanium: sapphire laser, will allow excitation throughout the red region of the electromagnetic spectrum (650-1000 nm). This will be valuable for studies of biological samples on gold substrates. Finally, new nonlinear techniques, such as hyper-Raman and surface-enhanced hyper-Raman scattering, will be possible using pulsed excitation. Acknowledgment This work was supported by the Divisions of Chemical Sciences (M.S. and T.M.C.) and Energy Biosciences (M.S.), Office of Basic Energy Sciences, U.S. Department of Energy; Consejo Superior de Investigaciones Cientificas, Spain (R.P.); and the National Institutes of Health (T.M.C., grant Number GM-35108). Ames Laboratory is operated for the U.S. Department of Energy by Iowa State University under Contract No. W-7405-Eng-82. SERI is a division of the Midwest Research Institute and operated for the U.S. Department of Energy under Contract No. DE-AC02-83CH-10093.
[5] S y n t h e s i s o f C a r o t e n o i d s S p e c i f i c a l l y L a b e l e d w i t h Isotopic Carbon and Tritium
By ARNOLD A. LIEBMAN, WALTER BURGER,
SATISH C. CHOUDHRY,
and
JOSEPH CUPANO
Introduction The chemistry used in the synthesis of carotenoids is quite extensive and has been periodically reviewed, l This literature reflects the expanding use of chiral reagents and other tools of modern organic synthesis and also the increasing use of isotopically labeled carotenoids in a variety of applit K. Bernhard, in "Carotenoids: Chemistry and Biology" (N. I. Krinsky, M. M. MathewsRoth, and R. F. Taylor, eds.), Proc. 8th Int. Syrup. Carotenoids, p. 337. Plenum, New York, 1989. METHODSIN ENZYMOLOGY,VOL.213
Copyright© 1992byAcademicPress,Inc. Allrightsofreproductionin anyformreserved.
