Structural Analysis ofBiological AliphaticCompounds Using Surface-Enhanced Fourier TransformRaman Spectroscopy

Takeshi HasegawaDepartment of Applied

Molecular Chemistry,College of Industrial

Technology,Nihon University,1-2-1- Izumi-cho,

Narashino,Chiba 275-8575,

Japan

Received 21 May 2003;revised 27 August 2003;

accepted 2 September 2003

Abstract: The surface-enhanced Raman scattering (SERS) technique for Fourier transform Ramanspectrometry is employed to reveal the chemical structure of biological aliphatic compoundsconsisting of folded, long aliphatic chains. The structural analysis is performed via the measure-ments of the accordion-vibration modes generated in the ordered, long aliphatic chain. The SERSspectra after subtraction of a background spectrum give segment lengths that are almost perfectlyconsistent with the chemical structures studied by mass spectrometry. The agreement of the SERSresults with those of mass spectrometry suggests the positions of kinks in the long hydrocarbonchain. The combination technique of SERS and mass spectrometry is useful to discuss the structureof folded, long biological lipids. © 2004 Wiley Periodicals, Inc. Biopolymers 73: 457–462, 2004

Keywords: structural analysis; biological aliphatic compounds; surface-enhanced Fourier trans-form Raman spectroscopy

INTRODUCTION

The structural analysis of biological compounds hasbeen a crucial matter in understanding the biologicalactivity of compounds. Biological compounds varyconsiderably in terms of their chemical structures andeven relatively simple biological compounds like al-iphatic derivatives are still difficult to analyze, such asin protein analysis. NMR,1 mass,2 and IR spectrom-etries3 have been used for the determination of chem-ical structures. In particular, electron-impact ioniza-tion mass spectrometry (EI-MS)4 is useful to ascertainthe unit-sequential structure of a line-shape compound

by breaking the linear chain. This is a great benefit ofMS, but other spectroscopic techniques are necessaryto know the structure of folded chains in situ that arenot determined by the chemical structure only. Forthis purpose, FTIR and NMR are commonly used forthe investigation of inter- or intramolecular interac-tions like hydrogen bonding. In addition, circular di-chroism (CD) spectrometry is also often employed inboth the UV–visible5 and IR6 regions to study thechanges in molecular folding.

We previously reported molecular-folding and mo-lecular-extension phenomena of �-mycolic acid thatwas extracted from a living cell envelope of acid-fast

Correspondence to: T. Hasegawa (t5hasega@cit.nihon-u.ac.jp).Contract grant sponsor: Ministry of Education, Science, Sports,

Culture, and Technology, Japan; contract grant number:413/14045267.Biopolymers, Vol. 73, 457–462 (2004)© 2004 Wiley Periodicals, Inc.

457

bacteria.7 Mycolic acids (1-alkyl branched 2-hydroxyfatty acids) have the common chemical structure pre-sented in Scheme 1, and the longer chain part is calledthe mero group.8 The mero group is varied in structureby changing the X and Y parts, and it is also knownthat the two parts can have cis and trans conformers.8

Another important parameter of mycolic acids is thesegment length represented by l, m, and n in thescheme. All these chemical parameters vary indepen-dently, which generates complicated characteristics ina biological system. In contrast, the shorter chain isknown to have only two patterns of length (k � 21 or23) in most mycolic acids.9,10

Our previous article7 revealed that �-mycolic acidin a monolayer had a folded form at a low surfacepressure whereas it was fully extended when a veryhigh surface pressure was applied, which was studiedby atomic force microscopy (AFM). This implies thata mycolic acid having a relatively simple chemicalstructure can have different higher order structures,which are influenced by external pressure. Judgingfrom the AFM images, it was suggested that the merogroup had one or two kinks to yield the folded form ata low surface pressure, and the kinks were present atthe cyclopropyl groups (located at X and Y, Scheme1). Nevertheless, it was difficult to confirm the exactkink positions, because the folding structure was sug-gested by considering only the AFM results throughthickness changes of the monolayer. A more struc-ture-sensitive analytical technique is required to re-veal the kink positions in the folded chain.

In the present study, Raman spectroscopy11–13 hasbeen employed to evaluate the segment length of theordered methylene-chain parts in the folded chain.Raman spectroscopy is a structure- and conformation-sensitive analytical tool, and it is widely used forstructural characterization of biological molecules. Inparticular, it is known that resonance Raman spectros-copy is powerful enough to analyze a specific part in

a complex macromolecule. In addition, Raman spec-troscopy has another useful function that enables us toevaluate the length of the methylene chain in anall-trans conformation.14 The key bands appear in avery low wavenumber region (ca. 20–300 cm�1) nearthe Rayleigh scattering, which arises from the accor-dion vibration of the all-trans methylene segments. In1967 Schaufele and Shimanouchi reported a simplerelationship between the segment length in the all-trans conformation and the band positions (�, cm�1),which is represented by the following equation14:

� � 2400/Nc (1)

where Nc is the number of methylene groups in theall-trans chain of interest.

It is historically known that polyethylene chainshave many repeating units that form the lamellarstructure, which is readily observed by the accordion-vibration bands in Raman spectra.14 In the lamellarstructure the chain bending generates shorter repeat-ing units than the entire chain. In this manner, theaccordion-vibration modes are observed for the “or-dered chain length” in a long chain.

Although using only the analysis of the accordion-vibration bands may be poor to confirm the foldingstructure, the combination analysis with MS would bepowerful. MS is an excellent technique to reveal thechemical structure by directly detecting the mass ofmolecular fragments. In particular, in the case ofmycolic acids, the cyclopropyl group is broken easilyby the ionization of the MS measurements, whichenables us to effectively analyze the saturated hydro-carbon parts other than the cyclopropyl groups. It isstill difficult, however, to discriminate folded andextended chains by MS only. If the chain foldingoccurs in a saturated hydrocarbon part, the chainlengths evaluated by the accordion-vibration analysiswould be different from the MS results. In otherwords, if the Raman and mass analyses were consis-tent with each other, it would strongly suggest that afolding point is located at a cyclopropyl group. My-colic acids are suggested to have a folded long chainthat comprises relatively ordered parts and kinks, andtherefore accordion-vibration analysis combined withMS data would be the most appropriate method forthe present structural study. It is expected that theordered parts will contribute to the accordion-vibra-tion bands, which will reveal the length of the parts.

A problem of measuring the accordion-vibrationmode is that the Raman cross section10 of this mode isvery low, and it is more difficult to measure minutesamples. Because this band is measured in the non-

SCHEME 1 The chemical structure of mycolic acids.

458 Hasegawa

resonance condition, the measurements are expectedto be very difficult. Thus, in the present study, thesurface-enhanced Raman scattering (SERS) tech-nique15 is employed to increase the scattering inten-sity of the vibrational mode. In order to do this, a goldaqueous colloid suspension16 is chosen for the en-hancement material after various trials. This approachstill has a problem that the mycolic acids are notinsoluble in an aqueous solution. In other words, it isdifficult to make the mycolic acids perfectly dispersein water so that they will be directly adsorbed on thegold particles. Fortunately, however, it is possible toprepare a stable suspension by mixing a tiny amountof thick chloroform solution of mycolic acid with thegold aqueous colloid.

With the aid of the surface enhancement by thegold colloid, the accordion-vibration modes arereadily measured by a normal FT-Raman spectrome-ter. FT-Raman spectroscopy has an experimental lim-itation that the excitation laser is limited to a Nd-YAGlaser (1064 nm). However, it has the advantage thatthe laser light does not raise the sample temperature toa great extent, which is suitable for analysis of bio-logical samples. FT-Raman spectroscopy is useful tomeasure the accordion-vibration modes with the aidof the SERS technique.

MATERIAL AND METHODS

The biological samples used in this study (�- and keto-mycolic acids) are from the Mycobacterium tuberculosis Kand M. kansasii 20-01 species, respectively, which wereextracted from a defatted freeze-dried cell mass by alkalinehydrolysis followed by methylation in a conventional man-ner.8,17 Both samples were kindly provided by Dr. MotokoWatanabe. Each chemical species was characterized byNMR and MS for our Raman analyses. The chemical struc-tures of the samples are represented by the general structurein Scheme 1. The analytical data suggested that �-mycolicacid comprised one chemical species, although it had somevariations in the segment lengths, whereas keto-mycolicacid comprised compounds with two different conforma-tions (Scheme 2).8

A sample of about 1 mg was dissolved in 50 �L ofchloroform in a microtube. Then 400 �L of gold-colloidalaqueous suspension (BB International, Cardiff, UK) wasadded to the microtube, and it was shaken by a Vortex mixerfor 1 min to yield a stable suspension. The suspension wastransferred into a short NMR tube (4-mm i.d. � 55-mmlength) made of quartz, and it was subjected to the FT-Raman measurements. The Raman spectrometer in thestudy was a Thermo-Electron (Madison, WI) Nicolet FT-Raman 960 that had a near-IR (NIR) laser (1064 nm) as theexcitation light source. The NIR light was focused in theNMR tube; and the scattered Raman light was collected by

a parabolic mirror, which led to a germanium detectorcooled by liquid nitrogen. The laser modulation frequencyby the interferometer was 8.9 kHz, and 2000 interferogramswere accumulated to improve the signal to noise ratio. TheRaman spectra on the Stokes side were recorded at a spec-tral resolution of 4 cm�1.

The measurements of the visible spectra for checking theplasmon absorption were made with a Beckman Coulter(Fullerton, CA) DU-600 UV–visible spectrometer at a con-stant room temperature of 25°C.

RESULTS AND DISCUSSION

A suspension of gold colloids adsorbed with �-my-colic acid was subjected to FT-Raman SERS spec-troscopy. The resulting spectrum is presented in Fig-ure 1. In the low wavenumber region for the analysisof the accordion vibration, only a broad curve withminute peaks appears, except for the band of chloro-form at 264 cm�1. In this manner we found that thedetection of accordion-vibration modes by nonreso-nance FT-Raman spectroscopy is difficult, even withthe SERS technique. For reference, an FT-Ramanspectrum of a gold-colloidal dispersion with no sam-ple is also plotted in the figure. The curve resemblesthe SERS spectrum of �-mycolic acid in shape, andno characteristic bands are found. A colloidal-disper-sion solution of gold colloids adsorbed with keto-mycolic acid was measured in the same manner byFT-Raman SERS spectroscopy. The raw keto spec-trum presented in Figure 1 also has a broad and

SCHEME 2 The possible chemical structures of mero-mycolic acids suggested by mass spectrometry.8

SERS FT-Raman Analysis of Aliphatic Compounds 459

similar shape, which makes no sense for chemicaldiscussion.

It is still difficult to measure nonresonance Ramanbands for accordion-vibration modes by using thesurface-enhancement technique. One of the difficul-ties of observation is that the scattering from the goldcolloid and water overlays on the weak Raman signal.In other words, the strong “background spectrum”interferes with the Raman measurements. Therefore,the background spectrum should be subtracted fromthe collected Raman spectra. In Raman spectroscopy,however, the observed intensity changes for eachmeasurement, which makes a simple subtraction anal-ysis difficult. In an absorption spectroscopy like IRspectroscopy, a background can be subtracted withoutproblem when the absorbance scale is used. However,a perfect subtraction of the background is generallydifficult for emission spectroscopy. When a spectrumwith minute intensity is extracted by the subtraction ofa strong spectrum, a proper subtraction is particularlydifficult. Therefore, in the present study the subtrac-tion was performed by applying a factor, so that thebaseline of the subtracted result would become flat.

The subtracted spectrum for �-mycolic acid is pre-sented in Figure 2. It was found by the subtractioncalculation that the minor bands clearly appear, aswell as the strong band due to chloroform. At leastfive bands were found (see Fig 2), which were attrib-uted to the accordion-vibration modes. Although theband position of an accordion-vibration mode isknown to obey the simple relationship [Eq. (1)] thatrelates to the length of a methylene segment, it isalready known that the band position does not exhibitperfect linearity when the methylene chain becomesshort and less than C16. Schaufele and Shimanouchi14

observed a number of Raman bands of n-paraffin for

the analysis of the longitudinal acoustic (LA) modesin finite polymethylene chains, and they found that therelation between band positions and methylene seg-ment lengths were formulated by a simple regressioncurve with a least-squares calculation. The regressioncurve is represented by Eq. (2).

� � 2495m

Nc� 5.9 � 103�m

Nc�2

� 6.3 � 104�m

Nc�3

(2)

where m indicates an index of overtone of the LAmode. Because we consider only normal modes (theaccordion vibration) in the present study, m is alwaysunity. With this equation, the observed band positionsare converted to segment lengths, which are summa-rized in Table I. Because the bands at about 211.1 and116.3 cm�1 are broad and weak, the segment lengthcan have two values as presented in the table. TheSERS spectrum suggests that there are methylenesegment lengths of 11 (or 12), 13, 14, 19, and 21 (or22). One may be concerned with the broadband atabout 160 cm�1, which corresponds to a segment

FIGURE 1 Raw FT-Raman SERS spectra in the region ofthe accordion vibration for �- and keto-mycolic acids and agold-colloid suspension (background).

FIGURE 2 An FT-Raman SERS spectrum of �-mycolicacid after subtraction of background.

Table I Accordion Vibration Bands and CalculatedSegment Lengths for �-Mycolic Acid

Band Position(cm�1)

Calc SegmentLength

Segmentsby MS

211.1(broad) 11.7(11or12) 11190.5 12.9

13187.0 13.2 13174.5 14.1 14132.2 18.6 19116.3(shoulder) 21.1(21or22) 22

460 Hasegawa

length of 15. In the present study, however, thisshoulder band is not discussed, because the peaklocation is unclear. In addition, MS analysis8 alsosuggested that some impurities are present because ofthe extraction of biological cells.

Watanabe et al.8 recently reported major methyl-ene segments in �-meromycolic acid that was pre-pared from a methyl mycolate fraction by pyrolysis.Each segment was analyzed by charge-remote frag-mentation MS18 after ionization by use of high-energycollision-induced dissociation. The notable results byMS are summarized in Table I. The results by MS areconsistent with the evaluated lengths by SERS. Thisagreement strongly suggests that the peaks in thesubtracted SERS spectrum truly arose from the accor-dion-vibration modes. Because the peaks were notavailable without SERS, the enhancement effect bythe use of adsorption on gold particles was found to bequite useful.

It should be noted that the SERS measurementswere readily performed with gold-colloid particleswith a diameter of 100 nm. When a smaller size ofgold colloid was used (e.g., 40 nm), almost no bandswere obtained even after background subtraction. Fig-ure 3 presents the visible spectra of the two gold-colloid suspensions. The absorption bands are arisenfrom the plasmon absorption in the gold particles.19 Itis clear that the 40-nm gold particles exhibit absorp-tion at a shorter wavelength, and the absorbance goesdown to zero below 800 nm. In contrast, the 100-nmgold particles exhibit a long tail toward the longerwavelength. Although the spectrometer has a wave-length window up to 800 nm, a difference of absor-bance was expected between the 40- and 100-nmparticles at 1064 nm, which is the excitation wave-length of the FT-Raman spectrometer. If we had an-other gold colloid with a diameter larger than 100 nm,it would be possible to have stronger SERS spectra.

The difference SERS spectrum presented in Figure4 was calculated for keto-mycolic acid as in Figure 2.This difference spectrum also has a flat baseline,which suggests that the subtraction of backgroundwas reasonably done. From this spectrum, five peaksare read, which are listed in Table II. The segmentlengths calculated from the band positions with theuse of Eq. (2), are also presented in Table II. Wa-tanabe et al.8 paid special attention to both the chem-ical structure and conformation of keto-meromycolicacid using MS and NMR, and they revealed that theketo-mycolic acid from M. kansasii 20-01 species hastwo types of molecular structures (see Scheme 2).They employed EI-MS to reveal the length of eachsegment (l, m, and n). Their evaluated lengths aresummarized in Table II for the two types.

Because types 1 and 2 have very similar chemicalstructures, it is not easy to spectroscopically discrim-inate the two compounds as presented in Table II.Fortunately, however, the table tells that type 1 lacksthe C14 segment that is found in type 2, which wasrevealed by MS. This apparent difference is useful todiscriminate the similar compounds. The results of thetype 2 molecule analyzed by SERS exhibit an almostidentical pattern to the MS results, including the C14

FIGURE 3 Visible absorption spectra of gold-colloid sus-pensions with diameters of (� � �) 40 and (—) 100 nm. FIGURE 4 An FT-Raman SERS spectrum of keto-my-

colic acid after subtraction of background.

Table II Accordion Vibration Bands and CalculatedSegment Lengths for Keto-Mycolic Acid

Band Position(cm�1)

Calc SegmentLength

Type-1by MS

Type-2by MS

174.3 14.1 14158.2 15.6 15, 16 16136.4 18.0 17, 18 17, 18119.7 20.4 20 20100.4 24.3 24 24

SERS FT-Raman Analysis of Aliphatic Compounds 461

segment. This suggests that the keto-mycolic acidmolecules adsorbed on gold particles are type 2 mero-residue rich.

Therefore, the analytical results by SERS are con-sistent with those by MS, which strongly suggests thatthe analytical technique measuring the accordion-vi-bration mode is useful for FT-Raman spectroscopy.The good agreement between SERS and MS experi-mentally confirms that chain folding is present atexactly the cyclopropyl or keto group that shouldbecome a kink, and bending does not happen in themethylene chains as expected.

CONCLUSION

The FT-Raman SERS technique was employed toanalyze the segment length of mycolic acids adsorbedon gold particles. The enhancement by SERS enabledus to measure according-vibration modes after sub-traction of a background spectrum. The segmentlengths estimated from the accordion-vibration bandswere found to be consistent with the results by MS,which suggested that the chain folding occurs at thesubstituted groups such as cyclopropyl and ketogroups in the meromycolate.

A discussion became possible by combining theanalytical results of both MS and SERS, which wasnot reported previously to the best of our knowledge.For the analysis of the chemical structure of biologicallipid compounds, MS is an essentially powerful tech-nique. In the present study, however, molecular fold-ing in biological molecules was first revealed by mea-suring the SERS spectra. The good agreement of theSERS results with MS also suggests that the aliphaticchain in the molecule has an ordered conformation,because the accordion-vibration modes appear onlywhen the methylene chain is in the all-trans confor-mation. This combined technique would therefore beuseful to analyze molecular folding for long-chainlipids.

The author greatly thanks Dr. Motoko Watanabe, who wasa faculty member of the Tokyo College of Pharmacy, forkindly providing him with the highly pure samples for

analytical data. The financial support of this work by aGrant in Aid for Scientific Research on Priority Areas (A),Dynamic Control of Strongly Correlated Soft Materials,from the Ministry of Education, Science, Sports, Culture,and Technology (Japan) is acknowledged.

REFERENCES

1. Hounsell, E. F. Prog Nucl Magn Reson Spectrosc 1995,27, 445–474.

2. Johnson, B. M.; Nikolic, D.; van Breemen, R. B. MassSpectrom Rev 2002, 21, 76–86.

3. Roeges, N. P. G. Guide to the Complete Interpretationof Infrared Spectra of Organic Structures; Wiley:Chichester, UK, 1994.

4. Morand, K. L.; Horning, S. R.; Cooks, R. G. Int J MassSpectrom Ion Process 1991, 105, 13–29.

5. Johnson, W. C., Jr. Annu Rev Phys Chem 1978, 29,93–114.

6. Nafie, L. A. Appl Spectrosc 2000, 54, 1634–1645.7. Hasegawa, T.; Nishijo, J.; Watanabe, M.; Funayama,

K.; Imae, T. Langmuir 2000, 16, 7325–7330.8. Watanabe, M.; Aoyagi, Y.; Mitome, H.; Fujita, T.;

Naoki, H.; Ridell, M.; Minnikin, D. E. Microbiology2002, 148, 1881–1902.

9. Goodfellow, M., Minnikin, D. L., Eds. Chemical Meth-ods in Bacterial Systematics; Academic: London, 1985.

10. Hasegawa, T.; Nishijo, J.; Umemura, J.; Theiß, W. JPhys Chem B 2001, 105, 11178–11185.

11. McCreery, R. L. Raman Spectroscopy for ChemicalAnalysis; Wiley–Interscience: New York, 2000.

12. Long, D. A. The Raman Effect; Wiley: Chichester, UK,2002.

13. Gremlich, H.-U.; Yan, B. Infrared and Raman Spec-troscopy of Biological Materials; Marcel Dekker: NewYork, 2001.

14. Schaufele, R. F.; Shimanouchi, T. J Phys Chem 1967,47, 3605–3610.

15. Koglin, E.; Kreisig, S. M.; Kopitzky, T. Prog ColloidPolym Sci 2002, 109, 232–243.

16. Jung, Y.; Tashiro, H.; Ikeda, T.; Ozaki, Y. Appl Spec-trosc 2001, 55, 394–398.

17. Jenkins, I. D.; Goren, M. B. Chem Phys Lipids 1986,41, 225–230.

18. Adams, J. Mass Spectrom Rev 1990, 9, 141–186.19. Maruyama, Y.; Ishikawa, M.; Futamata, M. Chem Lett

2001, 8, 834–835.

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