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IntroductionThe nonlinear, multiphoton process of two-photon absorption (2PA) has been gaining greater

interest among a number of multidisciplinary areas, particularly in the rapidly developing fields ofmultiphoton fluorescence imaging, optical data storage and switching, optical sensor protection,telecommunications, laser dyes, 3-D microfabrication, and photodynamic therapy (PDT).1-5 Thedemands of such applications exceed properties and reliabilities delivered by current organic mate-rials, underscoring the need for increasingly sophisticated nonlinear optical organic materials. Inparticular, compounds that undergo strong nonlinear, multiphoton absorption are beinginvestigated as materials for a wide variety of potential applications in areas ranging from opticalinformation storage, 3-D optical memories, biophotonics, materials science, and photochemistry.For example, it is projected that a multiphoton-based 3-D optical volumetric memory will provideup to three orders of magnitude more information in the same size enclosure relative to a 2-Doptical disk memory.1

Two-Photon Organic PhotochemistryAlthough a wealth of information regarding the understanding and applications of photochemi-

cal transformations has been obtained over the last half century, comparatively few studies of mul-tiphoton induced organic photochemistry have been reported. In fact, in most books of organicphotochemistry, there is scarcely a mention of simultaneous two-photon induced photochemistry. Abrief description can be found in Excited States in Organic Chemistry6 and Principles and Applications ofPhotochemistry.7 This said, the underlying principles of multi- or two-photon absorption are particu-larly meritorious, and warrant much further investigation (particularly with the advent of commer-cially available ultrafast pulsed lasers). In fact, the field of two-photon organic photochemistry is inits infancy, not unlike the field of single photon organic photochemistry 50 years ago before thepioneering work of George Hammond, Michael Kasha, Howard Zimmerman, and others.

Multiphoton absorption can be defined as simultaneous absorption of two or more photons viavirtual states in a medium. The theory of simultaneous absorption of two-photons was developedby Goeppert-Mayer in 1931,8 but remained mainly a conceptual curiosity because light sources werenot available of suitably high intensity. It was not until the early 1960s that the two-photon absorp-tion process was experimentally verified by Kaiser and Garrett9 who used pulsed lasers that pro-vided very high intensity. Normally, molecules are promoted from the ground state to an excitedstate through resonant single photon absorption. However, under appropriate conditions, this exci-tation can be accomplished by two-photon absorption (2PA). In 2PA, molecules exposed to highintensity light can undergo near simultaneous absorption of two longer wavelength photons medi-ated by a so-called “virtual state,” a state with no classical analog. The combined energy of the twophotons stimulates the molecule to a stable excited state. If the two photons are of the same energy(wavelength), the process is referred to as degenerate 2PA. On the other hand, if the two photons areof different energy (wavelength), the process is nondegenerate 2PA. When light passes throughmolecules, a virtual state may form, persisting for a very short duration (on the order of a few

Continued on page 3

Two-Photon Organic Photochemistry

Kevin D. Belfield, University of Central Florida

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From the Executive Director

D. C. Neckers, Executive Director, Center for Photochemical Sciences, Bowling Green State University

Recently my graduate students have really caught me off guard. They’ve presented me with papers and communi-cations from journals I’ve been subscribing to for almost as long as I’ve been in chemistry. That is no big deal. What isa big deal is that they’ve given me a photocopy of an interesting article a day or week before my hard copy arrives.That didn’t and couldn’t happen before because my hard copy of JACS or J. Org. Chem. always arrived before thelibrary’s copy. Now the on-line version is sometimes weeks in front of the arrival of the hard copies. My students, andI too, have much broader and more rapid literature access right at our fingertips.

The changes that the Internet created are amazing. For the first time in my life I can read the Jamestown New YorkPost-Journal, The New York Times, The Toledo Blade and the Chronicle of Higher Education at my desktop. For some of theseI have to be a subscriber. For most I do not. Our library maintains subscriptions to on-line versions of everything theycan. In addition to Chemical Abstracts and various patent data bases, my students and I can read almost any journal fitto print from our office desktop.

I guess one must ask what is the future of hard copy anything?

This is an important question we can, and continually do, ask about The Spectrum. Printing hard copy is expensiveand slow. Mailing hard copies is increasingly costly. When it comes to reference books, the old-fashioned setting ofcopy in type still happens but it’s on the way out. One wonders what is the future of series that take 9 to 12 monthsfrom the time of author submission to the date of publication. By the time review articles appear, several more papersin the field may have already been published.

I’d like to seek the input of our readers. The Internet has revolutionized the passing of information.

• How will this impact the way scientists obtain, seek and retrieve critical technical data and information?• Will paper and hard copy soon be obsolete?• Will literature searches be dated as a result? Or will they be more complete because more information is easier to

obtain?• Just how do you feel about Internet journal subscriptions?• What’s the future of the book publisher? More generally, of books?• How long will it be before everything we read we read on a screen in front of our face?

My questions are asked in all seriousness and I really want your input. Please use the following email address(dcnecke@bgnet.bgsu.edu) and let me know what you think. If we get a good sampling of responses from our readerswe’ll publish your thoughts in a future Spectrum. Thanks in advance.

In This Issue

Two-Photon Organic Photochemistry ................................................................................................................................. 1

From the Executive Director ................................................................................................................................................. 2

Novel Exploration of the Ability of Singlet Oxygen to Photobleach P. taeda Pulps ...................................................... 8

High Hopes for Hypericin ................................................................................................................................................... 15

Page 3 The Spectrum

Continued from page 1

femtoseconds). The 2PA can result only if a second photon arrives before the decay of the virtual state. Thus, 2PAinvolves the concerted interaction of both photons that combine their energies to produce an electronic excitationanalogous to that conventionally caused by a single photon of a correspondingly shorter wavelength.

Two-photon transitions can be described by two different mechanistic processes. For nonpolar molecules with alow energy, strongly absorbing state near the virtual level, only excited states that are forbidden by single photonselection dipole rules can be populated via two-photon absorption.1 The probability that this low energy state cancontribute to the virtual state is predicted by Heisenberg’s uncertainty principle with a virtual state lifetime approxi-mated as h/(4π∆E), where h is Planck’s constant and ∆Ε is the energy difference between the virtual and actual states.Using this equation, it is predicted that an allowed state can contribute to the formation of the virtual state for timetvirtual, which is equal to about h/(4π∆E) with the transition probability proportional to ∆µ2.1 In polar molecules, strong2PA can also occur by a different mechanism in which a large change in dipole moment (∆µ > 10 D) occurs uponexcitation of the ground state. Single photon allowed states can then be accessed via 2PA, and the virtual state lifetimeis proportional to ∆µ2, while the transition probability also scales with ∆µ2. In this case, both the ground and excitedstates can participate in the formation of the virtual state, which enhances 2PA.

One of the major features that distinguishes 2PA from single photon absorption, apart from selection rules, arisesby considering the rate of energy (light) absorption as a function of the incident intensity. In the single photon pro-cess, the rate of light absorption is directly proportional to the incident light intensity (dw/dt α I), and is independentof the rate of photons passing through the molecule. By contrast, the 2PA process depends on both spatial and tempo-ral overlap of the incident photons, therefore the rate of light absorption is proportional to the square of incident lightintensity (dw/dt α I2). Since the probability of a 2PA process is proportional to the square of the incident light inten-sity, photoexcitation is spatially confined to the focal volume. Such precise control of photoexcitation is intriguing,facilitating the development of new technologies, processes, and materials that require 3-D spatial resolution of physicalproperties, both static (permanent) and dynamic (reversible).

Applications of Two-Photon PhotochemistryThe quadratic, or nonlinear, dependence of two-photon absorption on the intensity of the incident light has sub-

stantial implications. For instance, in a medium containing one-photon absorbing chromophores, significant absorp-tion occurs along the path of a focused beam of suitable wavelength light, which can lead to out-of-focus excitation. Ina two-photon process, negligible absorption occurs except in the immediate vicinity of the focal volume of a lightbeam of appropriate energy. This allows spatial and radial resolution about the beam axis, which circumvents out-of-focus absorption and is the principle reason for two-photon fluorescence imaging.7 Using near-IR radiation toinduce fluorescence in biological samples which contain fluorophores, dynamic processes in living cells can also beimaged.3 The 2PA processes greatly reduce photobleaching of the fluorophore and photodamage of cells, which wouldoccur using conventional UV excitation sources. Two-photon scanning laser microscopy can be used as a nondestruc-tive diagnostic and evaluation tool to probe surfaces, interfaces, and fractures in materials. Recently, we have showndefects in substrates coated with fluorophore-labeled poly(methyl methacrylate) (PMMA) can be detected throughnondestructive 3-D optical sectioning using two-photon fluorescence microscopy.11 Two-photon fluorescent imagingprovides a well-defined three-dimensional image of the subsurface morphology and demonstrates the possibility ofusing this method as a tool in failure analysis of materials.

Two-Photon MicrofabricationIn contrast to the linear dependence of single photon absorption on incident light intensity in conventional

photopolymerization, the quadratic dependence of photoexcitation on light intensity in 2PA can be exploited to con-fine polymerization to the focal volume and achieve fabrication of microstructures via 3-D spatially resolved poly-merization.5,11,12 Near-IR two-photon induced polymerization was demonstrated using free-radical and cationicphotoinitiator systems. Recently, we reported the near-IR two-photon induced polymerization of acrylate monomersusing a commercially available photoinitiator system based on a visible light absorbing dye.5 Two-photon initiatedpolymerization was conducted at 775 nm via direct excitation of a commercially available dye (5,7-diiodo-3-butoxy-6-fluorone, H-Nu 470)13 in the presence of an arylamine, and (meth)acrylate monomer, resulting in an electrontransfer-free radical initiation process. The excitation wavelength was well beyond the linear absorption spectrum for

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5,7-diiodo-3-butoxy-6-fluorone (strong and weakabsorption maxima at 330 and 470 nm, respectively).The formation of polymeric microstructures with avariety of dimensions was accomplished with theH-NU 470 initiator/acrylate monomer and other ini-tiator systems (Figure 1a). In particular, arylketonephotoinitiators such as isopropylthioxanthone (ITX),benzoin methyl ether (BME), and an acylphosphineoxide (Irgacure 819, CIBA) were found to be effectiveinitiators (all have λmax < 400 nm), resulting in well-defined microstructures.

Cationic photoinitiated polymerization of epoxides,vinyl ethers, and methylenedioxolanes have receivedincreasing attention, due in large part to the oxygeninsensitivity of the cationic process. Commerciallyavailable diaryliodonium (CD-1012, Sartomer) andtriarylsulfonium (CD-1010, Sartomer) salts were foundto initiate polymerization of multifunctional epoxideand vinyl ether monomers, affording well-defined mi-crostructures (Figure 1b).11,12 Typical multifunctionalepoxide monomers investigated were a mixture of

poly(bisphenol A-co-epichlorohydrin), glycidyl end-capped and 3,4-epoxycyclohexylmethyl 3,4-epoxy-cyclohexanecarboxylate (K126, Sartomer) or Epon SU-8 (Shell) and K126 in 1:4 weight ratios, respectively, with 1 wt%of either the sulfonium or iodonium initiators.

Two-Photon PhotocycloadditionThe photochemical [π2s+π2s] cycloaddition reaction has proven useful in organic synthesis, resulting in formation

of two new carbon-carbon bonds and a maximum of four new stereogenic centers. The first [2+2] photocycloadditionreaction reported was the formation of carvone camphor on exposure of carvone to sunlight by Ciamician in 1908.14

Both intermolecular and intramolecular photocycloaddition reactions have received considerable attention over thepast several decades, emphasizing the importance of this synthetic transformation. The [2+2] photodimerization ofcinnamate-derivatized polymers faciliated the development of early photoresists that helped launch photolithogra-phy and the printed circuit board industry.

Under irradiation with UV light, psoralens, naturally occurring coumarin derivatives, are known to react withpyrimidine bases in DNA. Various physiological actions such as skin erythema, inactivation of DNA virus, and disor-ders in the development of sea urchin eggs fertilized with sperm have been attributed to this photoreaction.15 Theformation of interstrand crosslinking through C4-cycloaddition of 3,4- and 4’,5’-double bond with the 5,6-doublebond of the pyrimidine bases, especially thymine, in DNA has been correlated with biological effects of the photoexcitedfurocoumarin. The crosslinked DNA inhibits replication, causing cell death. This is the principle behind the use ofpsoralens in photodynamic cancer therapy.

The 2+2 photocycloaddition of 5,7-dimethoxycoumarin (DMC) was investigated as a model system for the studyof the photoinduced reaction of psoralens and pyrimidine bases in DNA.16 Unlike other coumarins, DMC only hasthe reactive pyrone 3,4-double bond which causes the photoreactivity. Thus, fewer photoproducts of DMC with thymineare formed due to this lack of bifunctionality. As a result, this led to easy isolation and analysis of photoproducts. Thestudy of photoaddition of DMC with thymine provided a good model for the photoreaction of psoralens with pyrim-idines, while the study of the photodimerization of DMC provided the fundamental understanding for this modelreaction.15 Single photon induced photodimerization of 5,7-dimethoxycoumarin was reported in the early 1990s.17,18

It was reported that photodimerization of DMC generated three different isomers: syn head-to-head, syn head-tail,and anti head-to-tail.

In order to compare two-photon induced photodimerization with the corresponding single-photon induced reac-tion, we chose the photodimerization of DMC. In anisole under long wavelength UV irradiation (350 nm broadband)two photodimers were observed (Scheme 1).19 Since the λmax of DMC is 320 nm, experiments were conducted by

Figure 1. Optical micrographs of microstructures created viatwo-photon polymerization of (a) an acrylate monomer(SR349) using the H-NU 470 initiating system (9 µm linewidth, 100 µm line spacing) and (b) a mixture ofpoly(bisphenol A-co-epichlorohydrin), glycidyl end-capped and3,4-epoxycyclohexylmethyl 3,4-epoxy-cyclohexanecarboxylate(K126, Sartomer) using CD-1012 (18 µm line width,progressively increasing line spacing beginning with 72 µm,from bottom to top).

Page 5 The Spectrum

irradiating DMC in anisole at 660 nm (110 fs pulse width). GC/MS analysis confirmed dimer formation, while HPLCanalysis allowed identification of the specific photodimers through comparison with authetic samples prepared viasingle-photon photolysis. It was found that the same two photodimers observed upon UV exposure of DMC wereformed after two-photon excitation, and the dimers were formed in nearly the same ratios. Further confirmation oftwo-photon absorption of DMC at 660 nm was secured by recording its two-photon upconverted fluorescencespectrum and observing the characteristic quadratic dependence of emission intensity as a function of incident(excitation) intensity.

Two-Photon PhotochromismPhotochemical transformations induced by 2PA have potential applications. Rentzepis et al. reported two-photon

induced photochromism of spiropyran derivatives using 1064 nm radiation.20,21 Analogous to single photon absorp-tion facilitated isomerization, the spiropyran underwent ring-opening isomerization to the zwitterionic coloredmerocyanine isomer. Like many spiropyrans, spirooxazine and fulgide-type compounds are known to undergophotoisomerization from a colorless to highly colored isomer.22 Unlike the spiropyrans, the thermally andphotochemically stable spirooxazine and fulgide-type compounds have been reported which underwent numeroussingle-photon photochemical isomerization (color) and reversion cycles without significant degradation.23 Opticaldata recording potentials as high as 100 million bits/cm2 have been reported for fulgide-type materials. In an effort todevelop a more photostable material for two-photon holographic imaging and information storage, fulgide 1(λmax = 385 nm) was chosen for study, particularly since its single-photon photochromic behavior is well established(Figure 2).24

Two-photon induced photochromism of 1 was demonstrated through determination of the kinetics of fs near-IR(775 nm) photoisomerization performed using a pump-probe experimental setup.11,25 As seen in Figure 3, formationof the fulgide photoisomer (2) was monitored as a function of time. Plots of absorbance at 585 nm (log I0/I) versus

O

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Figure 2. Photoisomerization of fulgide 1.

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Scheme 1

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time (s) were linear for the formation of the longer wavelength of the ring-closed photoisomer. The photoisomerizationrate constants thus obtained were 2.53 x 10-3 s-1 ± 0.3 x 10-3 s–1 and 6.99 x 10-3 s-1 ± 0.5 x 10-3 s-1 at irradiation intensitiesof 3.5 and 7.0 mW, respectively. As can be seen from the rate constants as a function of irradiant intensity, a near-quadratic dependency was observed for the photoisomerization of 1 as a function of intensity of the 775 nm fs pumpbeam, supportive of a two-photon induced process.

Preliminary 2-D interferometric recording was performed using a Mach-Zehnder interferometry setup using aClark CPA2001 775 nm fs laser as the irradiation source (Figure 4). Photoinduced changes were observed in theregions of high light intensity (bright interference fringes) in a thin film of poly(styrene)/fulgide 1 composite, demon-strating a proof of principle for effecting photochromic transformations in localized regions (Figure 4) as a model forholographic information storage.11,25 The dark lines in the image of Figure 4 result from high intensity bright fringe-induced photoisomerization of fulgide 1 (13 µm line width and 155 µm line spacing). Current efforts involve two-photon holographic volumetric recording in this material.

ConclusionThe vast number of advantages associated with nonlinear absorption-induced processes is fast propelling two-

photon absorbing materials and photochemical transformations to the forefront of several important fields. Suchmaterials and processes can be expected to be employed in 3-D volumetric optical recording, nondestructive 3-Dimaging, optical sensor protection, photodynamic therapy, and 3-D microfabrication. We can expect to witness break-throughs in years to come, due to the harnessing of spatially-resolved two-photon induced photochemical reactionsin organic materials.

AcknowledgmentsThe National Science Foundation (ECS-9970078, DMR9975773), the Research Corporation Cottrell College Science

Award (CC5051), donors of The Petroleum Research Fund of the American Chemical Society (35115-B4), and theNational Research Council are acknowledged for partial support of this work.

References1. Birge, R. R.; Parsons, B.; Song, Q. W.; Tallent, J. R. In Molecular Electronics; London, 1997; Chapter 15.2. Belfield, K. D.; Schafer, K. J.; Mourad, W.; Reinhardt, B. A. J. Org. Chem. 2000, 65, 4475.3. Herman, B.; Wang, X. F.; Wodniki, P.; Perisamy, A.; Mahajan, N.; Bery, G.; Gordon, G. In Applied Fluorescence in

Chemistry, Biology, and Medicine; Springer: New York, 1999; pp 496-500.

M

S

MBS

BSM = Mirrors

S = Sample

BS = Beam splitter

CPA2001 Laser

Figure 4. Schematic of a Mach-Zehnder interferometer for 2-D recording viatwo-photon photochromism (top left). Dark lines in image (right) result from highintensity bright fringe-induced photoisomerization of fulgide in a polystyrene film(13 µm line width and 155 µm line spacing, intensity = 120 mW, λ = 775 nm, laserexposure time = 0.5 to 3 min, output beam angle = 2°).

4. Belfield, K. D.; Schafer, K. J.; Hagan, D. J.; Van Stryland, E. W., Negres, R. A. Org. Lett. 1999, 1, 1575.5. Belfield, K. D.; Ren, X. B.; Van Stryland, E. W.; Hagan, D. J.; Dubikovski, V.; Meisak, E. J. J. Am Chem. Soc. 2000, 122,

1217.6. Barltrop, J. A.; Coyle, J. D. In Excited States in Organic Chemistry; Wiley: New York, 1975; p 46.7. Wayne, R. P. In Principles and Applications of Photochemistry; Oxford University Press: New York, 1988; pp 58-63.8. Goeppert-Mayer, M. Ann. Physik 1931, 9, 273.9. Kershaw, S. In Characterization Techniques and Tabulations for Organic Nonlinear Optical Materials; New York, 1998;

Chapter 7.10. Denk, W.; Strickler, J. H.; Webb, W. W. Science 1990, 248, 73.11. Belfield, K. D.; Schafer, K. J.; Liu, Y.; Liu, J.; Ren, X.; Van Stryland, E. W. J. Phys. Org. Chem. 2000, 13, 837.12. Belfield, K. D.; Ren, X.; Liu, J.; Van Stryland, E. W. RETECH Conference Proceedings, 12th International Conference on

Photopolymers 2001, in press.13. The fluorone dye, 5,7-diiodo-3-butoxy-6-fluorone (H-NU 470) and N,N-dimethyl-2,6-diisopropylaniline, were

obtained from Spectra Group Limited, Inc.14. Crimmins, M. T.; Reinhold, T. Org. React. 1993, 44, 297.15. Sang, C. S.; Kyu, H. C. Photochem. Photobiol. 1979, 30, 349.16. Sang, C. S.; Hun, Y. K.; Dae, Y. C. Photochem. Photobiol. 1981, 34, 177.17. Sang, C. S.; Bong, M. J.; Young, H. P. Bull. Korean Chem. Soc. 1992, 13, 684.18. Sang, C. S.; Kwan, Y. C.; Pill-Soon, S. Photochem. Photobiol. 1978, 27, 25.19. Liu, Y. M.S. Thesis, University of Central Florida, 2000.20. Parthenopoulos, D. A.; Rentzepis, P. M. Science 1989, 245, 843.21. Dvornikov, A. S.; Rentzepis, P. M. Opt. Commun. 1995, 119, 341.22. Photochromism Molecules and Systems; Durr, H.; Bouas-Laurent, H., Eds.; Elsevier: New York, 1990; Chapters 8-10.23. Heller, H. G. Fulgides and Related Systems. In CRC Handbook of Photochemistry and Photobiology; CRC Press: Boca

Raton, FL, 1995; p 181.24. Janicki, S. Z.; Schuster, G. B. J. Am. Chem. Soc. 1995, 117, 8524.25. Belfield, K. D.; Hagan, D. J.; Liu, Y.; Negres, R. A.; Fan, M.; Hernandez, F. E. Proc. SPIE - Int. Soc. Opt. Eng. 2000,

4104, 15.

About the AuthorKevin D. Belfield received a Ph.D. in chemistry from Syracuse University in 1988, where he studied the kinetics

and mechanism of thermal rearrangements of organic molecules with John E. Baldwin. As a postdoctoral researcherat SUNY College of Environmental Science and Forestry with Israel Cabasso, he worked in the area of synthesis andcharacterization of functionalized polymers and synthetic polymer membranes. He then pursued postdoctoral re-search in mechanistic organic chemistry with William von E. Doering at Harvard University, utilizing photochemicalreactions and determining radical stabilization energies of polyenyl radicals through spectroscopically-aided kineticstudies. He joined the faculty of the Chemistry Department at the University of Detroit Mercy in 1992. In 1998, hemoved to the University of Central Florida in 1998 to establish a program in the synthesis and characterization ofnonlinear optical organic materials and polymers. He is an Associate Professor in the Department of Chemistry,School of Optics/Center for Research and Education in Optics and Lasers (CREOL), and the Department of Mechani-cal, Materials, and Aerospace Engineering. His address is Department of Chemistry and CREOL/School of Optics,University of Central Florida, P.O. Box 162366, Orlando, FL 32816-2366; e-mail: kbelfiel@mail.ucf.edu.

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Novel Exploration of the Ability of Singlet Oxygen toPhotobleach P. taeda Pulps

Lucian A. Lucia, Georgia Institute of Technology, and Ki-Oh Hwang, Cargill, Inc.

IntroductionPulp and paper manufacture is a central part of a global, diverse, and dynamic industry that contributes to

products that in general economists equate with increased quality of life, that is, tissue, boxes and packaging, boardproducts, paper and writing materials, lumber/wood, veneer, resins, and even surplus energy.1 The commercialnature of the industry typically garners a high ranking in productivity and contribution to the US national GNP withan output of 87 million metric tons (30% world total) of pulp and paper products (excluding lumber products) at theend of the last century.2 Despite its production efficiency and contribution to state economies, the industry reliesheavily on capital expenditures that unfortunately mitigate overall annual growth to several percent. New, funda-mental breakthroughs in its technological operations are therefore required to provide an avenue for future improvedgrowth. The partnership of the Institute of Paper Science and Technology (IPST) with industry addresses the need forbasic chemistry knowledge in the industry. The partnership is a academic/industrial relationship 60 years in themaking before the creation of the NSF Science and Technology Centers (STCs) established in 1989 to “help maintainUS preeminence in science and technology and ensure the requisite pool of scientists with the quality and breadth ofexperience required to meet the changing needs of science and society—ingredients essential to successfuleconomic competitiveness”.3

IPST is a private graduate school that is academically allied with the Georgia Institute of Technology and remainsthe principal research arm of the US and currently the global pulp and paper industry. Since many of the capitalexpenses incurred by the industry are associated with increasing the efficiency of the pulping and bleaching chemicalreactions (the core of the industry processes), the work at IPST described in this paper will focus on chemical bleach-ing reactions. The generation of high-value products in the industry begins with the incipient pulping and bleaching(a fine tuning of pulping) reactions. Pulping is a process practiced globally to remove the “lignin” in woody tissuefrom the valuable carbohydrates (cellulose and hemicelluloses) and is dominated by the well-known kraft processand its sundry modifications.4 Figure 1 displays the three essential building blocks or propanoid phenol units, C9,“monomer” units of lignin that are assembled via an enzyme-mediated radical coupling process (lignification) thatgenerates “lignin.” Lignin (from Latin, lignum, wood) is a three-dimensional matrix of these units condensed at differ-ent C-C bond sites as shown in Figure 2.

In general, the bulk (greater than 50%) of lignin linkagesin softwoods (such as pine and spruce) are β-O-4 (the β car-bon of one propyl monomer is connected to the p-hydroxyoxygen of another monomer). The kraft pulping processconsists of hydrolyzing the linkages between monomers bythe introduction of a highly basic hydrosulfide solution athigh temperatures and pressures as shown in Figure 3. Thebasic conditions deprotonate the phenol hydroxy groupsand resonate the phenoxy anion to form quinone methides(QM) that are susceptible to nucleophilic attack byhydrosulfide anion. The hydrosulfide anion attacks the α-carbon of a QM to reform the phenolate anion and subse-quently attacks the β-carbon to form a thirane structure withthe concomitant expulsion of the β group (pulping concept).

Bleaching is an extension of pulping and is designed toremove the residual lignin that cannot be effectively re-moved in pulping without complete degradation of the

Figure 1. The three common building blocks of lignin arep-coumaryl alcohol, sinapyl alcohol, and coniferylalcohol, and are simple derivatives of the structureshown above.

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Page 9 The Spectrum

carbohydrates. A breakthrough in bleaching that we have made recently was determining that singlet oxygen couldeffectively be employed to oxidatively attack the last vestiges of lignin remaining in kraft softwoods, specificallySouthern pine, Pinus taeda, having lignin contents of 4%.

The Applicability of Singlet Oxygen to SelectivelyPhotobleach the Lignin in P. taeda

Singlet oxygen can be generated via a step-wise pho-tochemical process. In general, the photochemical pro-cess is very facile and requires a photosensitizing agentto transfer the excitation energy to the triplet groundstate of oxygen (3O2). The photochemical reactionscheme is shown in the following equation:

Sensitizer + hν → Sensitizer* + 3O2 → Sensitizer + 1O2

The focus of this work was to generate singlet oxygenin situ and allow it to react with residual lignin in pulpsamples without deleteriously affecting the native car-bohydrate component. Past work in the realm of“photobleaching” has dealt with model compoundsand methylene blue as the photosensitizing com-pound.5,6 These schemes, however, were not predictiveof the behavior of singlet oxygen in more complex P.taeda matrices (that include carbohydrates and organic

Figure 2. Shown at the left is a modernday depiction of the currently acceptedmacrostructure of lignin in wood asconstructed from various spectroscopicand chemical analyses of various ligninfunctionalities of wood. Unfortunately, atrue depiction of native lignin is well nighimpossible given the difficulty ofextracting a “pure” sample and itsnoncrystalline, highly amorphous nature.

Figure 3. The sulfidolytic cleavage of β-aryl ether bonds inpropyl phenol units as part of the overall chemical process ofkraft pulping.

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fatty acids) and did not employ high quantum yieldphotosensitizing agents. We employed Rose Bengal(RB), whose structure is shown in Figure 4, that has aquantum yield of 0.76 for singlet oxygen production inwater.7 Interestingly, RB aggregated easily with the lig-nin functionalities in the pulp as evidenced by its par-titioning between the pulp and aqueous phase as a func-tion of lignin content (the pulp uptake of RB was greatlyenhanced with increasing amounts of lignin in thepulp). Since singlet oxygen is highly electrophilic, elec-tron transfer reactions with lignin were highlyprobable, given free energies of several hundred milli-volts. Presumably, π-π stacking interactions between thepolyaromatic RB sensitizer and the aryl lignin unitsenhanced the tendency of RB to associate withthe lignin.

The oxidative ability of 1O2 is also known to be af-fected by a variety of factors, including pH and con-centration of hydroxyl anions. Work in the bleachingof textiles by 1O2 demonstrated that the optimal pH for1O2

8 production is approximately 9-10. We determinedthat an untreated pulp sample that is allowed to stand in deionized water (pH = 6.0) for long periods of time (> 200hours) would achieve a steady state pH of 9.2, roughly what is required for optimal 1O2 activity. Unfortunately, watertends to quench 1O2 via coupled vibrational interactions causing the lifetime window to be on the order of severalmicroseconds. Nonetheless, the close proximity of the sensitizer to the lignin was sufficient to provide ample oppor-

tunity for the photochemistry. Although singlet oxy-gen reactions with lignin species are favorable as sub-stantiated by past literature reports of lignin modelcompounds,9,10 lignin requires an alkaline environmentto encourage its dissolution. We found that the best pHfor removal of lignin was approximately 10. We foundthat we could photochemically remove approximately85% of the lignin in a sample having a starting lignincontent of about 3%. Figure 6 illustrates a generic reac-tion model for the manner in which singlet oxygencauses photobleaching.

Reaction Conditions for PhotochemistryAll work was done in deionized water that had a

concentration of 1-2% mass/mass of P. taeda pulp/wa-ter while the homogeneity of the suspension was main-tained by thorough mixing. Figure 5 provides an illus-tration of the setup that was employed. The system wasdesigned to capture the light emitted from the lamp,while the suspension was maintained at 45 ºC for theduration of the experiment. We used a 0.5% mass/massconcentration of RB relative to pulp, but 17% relativeto the amount of lignin in the starting sample (0.7mmoles per 100 g of pulp sample).

Based on an average Mw of 50 kD for lignin, the levelof RB added corresponds to approximately 10 mol-ecules of RB/C9 lignin monomer unit. Although this

Figure 4. The chemical structure of Rose Bengal, thephotosensitizer that was employed in our photobleachingschemes.

Figure 5. The photochemical apparatus that was employed forphotobleaching of the softwood pulp samples. A 400 WHanovia lamp was employed, while the sample was mixed andthe internal temperature of the 1% pulp sample suspensionwas maintained at 45 °C with an ice bath.

Page 11 The Spectrum

appears high, two factors competing against itsphotobleaching activity are the overwhelming excessof carbohydrates in the P. taeda sample (97% of sample)and the depletion of RB by singlet oxygen.

Photobleaching Provesto be a Promising Bleaching Process

Several fundamental physical and chemical featureswere monitored to assess the ability of singlet oxygento successfully bleach P. taeda samples: (1) reduction intotal lignin content (photobleaching), (2) reduction inthe overall chromophoric content of P. taeda (the “col-ored” lignin forms) and (3) impact on carbohydrates.

The photobleaching results are displayed inFigure 7. The effects of lignin leaching as a function ofbase content and heat are investigated separately with0.1 N NaOH concentration and 45 ºC (temperatureachieved during the irradiation). Past work in ligninleaching (autodelignification based on residual NaOHcontent and temperature) suggests that 20% leachingat this temperature is not unusual,11 especially at pro-tracted times after almost complete residual NaOH con-

sumption. Interestingly, we observed 36% lignin removal even in the absence of the photosensitizer, which realisti-cally is approximately 16% after accounting for lignin leaching. Delignification in the absence of RB is not unexpectedsince carbonyl groups in lignin (very small native mole-percentage, perhaps less than 1 percent) can photosensitizethe formation of singlet oxygen.12

RB provided significant delignification that wasenhanced in an alkaline environment. Lignin removalup to 90% was surprising, but not unexpectedgiven the reactive nature of singlet oxygen and thecomplete absence of RB after the irradiation fromoxidation reactions.

A more fundamental investigation into the chemi-cal basis for the effect of singlet oxygen on lignin wasobtained by systematically extracting the lignin frompulp using published methods13 and reacting it withsinglet oxygen generated chemically (hypochlorite/hydrogen peroxide) at 10% yield. We did not employthe lamp to avoid the direct photolysis of the ligninsamples from the high intensity irradiation. We discov-ered that the carboxylic acid content of lignin increasedby almost 40% (from an original value of 0.28 mmolCOOH groups/g lignin), while the 5,5-aromatic ligninforms (condensed monomer structures) decreased byabout 5%. The increase in acid groups helps to accountfor increased dissolution in alkaline environments,while the condensed lignin concentration decrease isprobably a likely result of more favorable electron trans-fer reactions. In fact, pulps that had a significantlyhigher condensed lignin content were significantlymore reactive toward singlet oxygen. This latter find-ing was supported during our studies of the decrease

Figure 7. The results of irradiating typical kraft pulped P. taedapulp with RB and a 400 W Hanovia medium-pressure mercurylamp after 22 hours.

Figure 6. Reaction mechanism describing the singlet oxygenoxidation of lignin-like monomers. The highly oxidizedaromatic units are susceptible to further oxidation, includingan intramolecular superoxide Michael reaction (afterdeprotonation of the terminal hydroperoxyl group).

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in chromophoric content (lignin absorbance) of thepulp samples.

For example, we took P. taeda samples that had approxi-mately 30-50% higher condensed 5,5-aromatic lignin con-tents (pulps that had been treated with high oxygen pres-sures to encourage radical coupling reactions), and foundsignificantly increased delignification kinetics in thesesamples. Figure 8 clearly demonstrates the accelerated ki-netics for the enriched condensed lignin sample over thesample containing “normal” lignin. Clearly, the enrich-ment in the condensed lignin structures exerts a profoundinfluence on the manner in which the singlet oxygen be-haves. This may be a function of the more favorable elec-trochemistry as indicated earlier in which the condensedstructures have lower oxidation potentials from the re-sultant conjugation (coupling of aromatic units).

In general, singlet oxygen degrades native pulp lignin.Figure 9 provides a UV/VIS spectrum of the absorbancedifference in a lignin sample from a standard kraft pulpdegraded by singlet oxygen that is dissolved in 1/1/0.1

dioxane/water/0.1 N HCl (0.09 mg/L). The difference of approximately 0.01 absorbance units although not appar-ently significant is dramatically reflected in the color of the lignin samples. The singlet oxygen treated sample is lightin color in comparison to the nontreated dark brown colored sample.

Finally, the issue of the carbohydrate chemistry is most important to the ultimate success of singlet oxygen as aviable bleaching chemical. The damage sustained by the carbohydrates in pulp samples is reflected by the intrinsicviscosity, a measure of the integrity of the cellulose polymer in the sample. Normally, cellulose chain lengths in kraftpulps are approximately 10,000 repeat glucose units that correspond roughly to a CED (cupriethylene diamine) vis-cosity of about 25-35 centipoise. The object of any pulping or bleaching technology is to maintain the natural viscosityto preserve the physical characteristics of the sample. Not surprisingly, although singlet oxygen attacks electrophilicstructures with a high specificity, the byproducts of the reaction are not entirely discriminatory in their reactivity. Forexample, as shown in Figure 6, superoxide is a direct byproduct of the electrophilic aromatic attack and has beenshown to disproportionate to various oxygen species such as hydroperoxyl radical and hydroxyl radical in the pres-ence of metals.14 The latter oxygen species are responsible for carbohydrate degradation in P. taeda and have been

controlled to some extent in this lab by the use of MgSO4,that is generally thought to help complex various activemetals such as Mn+2 and Fe+2 in a colloidal “solution” toprevent Fenton chemistry from occurring. Figure 10 dis-plays the initial high selectivity displayed by the chemi-cal bleaching system, not unlike what would be expected;yet, at 60% delignification, a steep drop in the integrity ofthe sample is observed. This peculiar finding may reflectthe increased reactivity competition between the ligninand the carbohydrates for the local oxygen speciation.

We tested the ability of MsSO4 to reduce the level ofhydroxy radicals from hydrogen peroxide breakdown andthereby maintain the system viscosity. Figure 11 shows atypical profile of the sample viscosity as a function of time.We noticed, for example, that the use of bubbled oxygenin the reactor cavity increased the rate of carbohydratedegradation, presumable because of increased reactivespecie concentration. On the other hand, including theMg+2 salt reduces this likelihood dramatically, but does

Figure 8. A plot of the changes in k/s, the absorption/scattering coefficient ratio, versus irradiation time for anormal lignin sample versus the same sample that isartificially enriched in condensed lignin units.

Figure 9. UV/VIS spectra of lignin dissolved in dioxanebefore and after singlet oxygen reaction.

not staunch it altogether, presumably because of local species competition at the lower lignin concentrations. The 60%delignification mark is reached at about 10 hours at which point the viscosity is well preserved; however, even withMg+2, the selectivity is not absolutely preserved. Further studies with more condensed lignin structures at lowerconcentrations (< 2%) suggest that despite the preferential reactivity of singlet oxygen with lignin, the ensuing radi-cals from the superoxide speciation are even more indiscriminatory in their reactivity and have greater opportunityto react with cellulose since the lignin levels are so much lower. Strangely enough, this indiscriminatory reactivity isbeneficial for reducing the chromophoric content significantly (by approximately 50%) since the activity of the radi-cals is not mitigated.

Concluding RemarksThe studies described above have been part of a new study to explain the ability of reactive oxygen species such as

singlet oxygen to selectively degrade the lignin in wood pulp. Our work above, for example, has demonstrated thatdespite the initial kinetics of the singlet oxygen-lignin reaction, reaction selectivity is a function of the oxygen speciesin the environment and reaction thermodynamics. Although cellulose is five to six times slower to react with hydroxyradicals than lignin,15 its proclivity to react is exponentially increased when the lignin concentration in the environ-ment decreases by 60%. Nonetheless, these studies represent the first known study to examine the photochemicalability of singlet oxygen to selectively degrade the lignin in P. taeda.

References1. Smook, G. A. Handbook for Pulp & Paper Technologists, 2nd ed.; Angus Wilde: Vancouver, 1992; pp 1-7.2. http://www.aifq.qc.ca/english/stats/papers01.html3. Committee on Science, Engineering, and Public Policy. An Assessment of the National Foundation’s Science and

Technology Centers Program; National Academy Press: Washington, DC, 1996.4. 1998 TAPPI Proceedings. Breaking the Pulp Yield Barrier Symposium; TAPPI Press: Atlanta, 1998.5. Crestinin, C.; D’Auria, M. Photodegradation of Lignin. The Role of Singlet Oxygen. J. Photochem. Photobio., A

1996, 101, 69-73.6. Ruggiero, R.; Machado, A. E. H.; Castellan, A.; Grelier, S. Photoreactivity of Lignin Model Compounds in the

Photobleaching of Chemical Pulps 1. Irradiation of 1-(3,4-Dimethoxyphenyl)-2-(3’-Methoxyphenoxy)-1,3-Dihydropropane in the Presence of Singlet Oxygen Sensitizer or Hydrogen Peroxide in Basic MethanolSolution. J. Photochem. Photobio., A 1997, 110, 91-97.

Page 13 The Spectrum

Figure 10. A plot of the intrinsic viscosity changes of thepulp samples as a function of delignification by applicationof singlet oxygen over 22 hours.

Figure 11. A plot of the sample intrinsic viscosity is shownas a function of time, oxygen sparging, and use of a metalcomplexing system.

7. Foote, C. S.; Clennan, E. L. Properties and Reactions of Singlet Dioxygen. In Active Oxygen in Chemistry; Foote,C. S. et al., Eds.; Blackie Academic & Professional: London, 1995; Chapter 4.

8. Thompson, K. The Role of Singlet Oxygen in the Bleaching of Cotton. Ph.D. Thesis, Georgia Institute ofTechnology, 1996.

9. Crestini, C.; D’Auria, M. Singlet Oxygen in the Photodegradation of Lignin Models. Tetrahedron 1997, 53,7877-7888.

10. Machado, E. H. A.; Gomes, A. J.; Campos, C. M. F.; Terrones, M. G. H.; Perez, D. S.; Ruggiero, R.; Castellan, A.Photoreactivity of Lignin Model Compounds in the Photobleaching of Chemical Pulps 2. Study of theDegradation of 4-hydroxy-3-methoxy-benzaldehyde and Two Lignin Fragments Induced by Singlet Oxygen. J. Photochem. Photobio., A 1997, 110, 99-106.

11. Li, J.; MacLeod, J. M. Alkaline Leaching of Kraft Pulps for Lignin Removal. J. Pulp Pap. Sci. 1993, 2, 85-92.12. Nimz, H. H.; Turznik, G. Reactions of Lignin with Singlet Oxygen. I. Oxidation of Monomeric and Dimeric

Model Compounds with Sodium Hypochlorite-Hydrogen Peroxide. Cell. Chem. Technol. 1980, 14, 727-742.13. Lucia, L. A.; Goodell, M. M.; Chakar, F. S.; Ragauskas, A. J. Breaking the Oxygen Delignification Barrier: Lignin

Reactivity and Inactivity. In Oxidative Delignification Chemistry: Fundamentals and Catalysis; Argyropoulos, D. S.,Ed.; ACS Symposium Series, Chapter 5.

14. Gierer, J.; Reitberger, T.; Yang, E. The Role of Superoxide Anion Radical O2- in Delignification. 7th International

Symposium on Wood and Pulping Chemistry, 1993; Vol. 1, pp 448-452.15. Ek, M.; Gierer, J.; Jansbo, K. Study on the Selectivity of Bleaching with Oxygen-Containing Species.

Holzforschung 1989, 43, 391-396.

About the AuthorsLucian Lucia is currently an assistant professor of chemistry at the Institute of Paper Science and Technology

(IPST) and maintains an active adjunct status at the Georgia Institute of Technology. Dr. Lucia obtained his Ph.D.degree in 1996 at the University of Florida and proceeded to work at the NSF Center for Photoinduced Charge Trans-fer at the University of Rochester as a postdoctoral fellow under Professor David Whitten. Following the postdoctoralfellowship, he went to Japan to briefly study in the laboratories of Professor Haruo Inoue, whereupon he accepted hiscurrent position at IPST. His group maintains an active interest in exploring the chemistry of oxygen and its relatedspecies with lignin and other biopolymeric materials. Several of Dr. Lucia’s interests are gardening, puzzle solving,his three children and wife (Cecilia, Claudia, Joshua, and Debbie), and teaching. He may be reached at 678-290-1273or Lucian.Lucia@ipst.gatech.edu.

Ki-Oh Hwang is currently a senior chemist at Cargill, Inc. in Eddyville, Iowa. Dr. Hwang obtained his Ph.D. degreein chemistry in 1993 at the University of Nevada, Reno. He worked at the University of Washington, University ofNevada, and Institute of Paper Science and Technology as a postdoctoral fellow before he joined Cargill, Inc. Hisresearch area includes synthesis, analysis, bench-top process development, and photochemistry. He may be reachedat 641-672-0895 or khwang@lisco.com.

The Spectrum Page 14

Copyright 2001 by the Center for Photochemical SciencesThe Spectrum is a quarterly publication of the Center forPhotochemical Sciences, Bowling Green State University,Bowling Green, OH 43403.Phone 419-372-2033 Fax 419-372-0366Email photochemical@listproc.bgsu.eduWWW http://www.bgsu.edu/departments/photochem/

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Articles submitted to The Spectrum will appear at the discretion ofthe editorial staff as space is available.

High Hopes for Hypericin

Peter A. Ensminger, Syracuse, New YorkThis article is excerpted from Life Under the Sun, by Peter A. Ensminger.

© 2001 Yale University Press, New Haven, Connecticut. Printed by permission.

Saint John’s wort has been in the news quite a lot in recent years. In the late 1980s, several studies showed that redpigments derived from this herb—hypericin and pseudo-hypericin— inactivated or blocked the reproduction of cer-tain retroviruses in mice. This led to speculation that these compounds might be effective against human retroviruses,including the human immunodeficiency virus (HIV), the virus associated with AIDS.1 Indeed, subsequent pre-clinical studies showed that hypericin could destroy the equine infectious anemia virus and the related human immu-nodeficiency virus.2 By the start of 2001, no clinical studies have demonstrated that hypericin or Saint John’s wort isan effective treatment for AIDS, although research in this area continues.3

Another line of research has shown that Saint John’s wort may effectively treat mild to moderate depression andseasonal affective disorder, while causing fewer side effects than certain standard anti-depressant drugs.4 Many stud-ies have examined the biochemical basis of this anti-depressant effect. Surprisingly, crude extracts of the plant onlyexhibit weak activity in biochemical assays related to the neurochemical mechanisms of widely used antidepressants,such as inhibitors of monoamine oxidase (nardil and parnate) and serotonin reuptake (prozac, zoloft, and paxil).5

Crude extracts of Saint John’s wort have shown affinity for nerve cell receptors that normally bind to gamma-aminobutyric acid (GABA), an important neurotransmitter in the brain.6 Barbiturates, benzodiazepines (Valium andXanax), and alcohol all exert their profound effects on the central nervous system by binding to GABA receptors.7 Atpresent, it must be concluded that the mechanism by which Saint John’s wort alleviates depression is unknown. It ispossible that the combined action of numerous mechanisms accounts for the overall effect. Future laboratory studiesmust be directed toward determination of the active ingredient(s) and its mechanism of action, and clinical studiesshould employ more standardized doses, longer lasting trials, and comparisons with more modern anti-depressants.8

Study of the myriad of possible therapeutic effects of Saint John’s wort and hypericin continues,9 as it has at leastsince the time of John Gerard, a sixteenth century English herbalist. This gives many researchers high hopes for Saint

John’s wort. At the same time, photobiologists have beenstudying the function of naturally-occurring hypericin-like pigments in two free-living microorganisms, Stentorcoerulus (see Figure 1) and Blepharisma japonicum (seeFigure 2). The hypericin-like pigments in these microor-ganisms, known as “stentorin” and “blepharismin”, arephotoreceptive pigments that control cellular locomotion(see Figure 3). Understanding the mechanism by whichthese pigments control cellular movements may clarifythe mechanisms by which hypericins act as anti-viralagents and Saint John’s wort as an antidepressant. At amore fundamental level, study of the light-induced move-ment responses of Stentor and Blepharisma may provide amore basic understanding of the chemistry of hypericins,a truly unique class of biological pigments.

Biology of the CiliatesStentor coerulus and Blepharisma japonicum are classified

as Ciliates, a taxonomic group of single-celled, non-photosynthetic organisms that are covered with cilia, shortwhip-like appendages that beat back and forth to propel

Figure 1. Negative phototaxis in Stentor.Herbert S. Jennings, an early researcher of behavior inciliates and other single-celled organisms, made this linedrawing of the view through a microscope of Stentorcoerulus. The illustration shows that cells move aboutrandomly in darkness, but when the anterior end of a cellenters a lighted region (1), it turns away (2) and swims backtoward darkness (3 and 4). The lighted area remains emptybecause of this negative phototactic response.

Page 15 The Spectrum

The Spectrum Page 16

them through the water or sweep food into their mouth-like openings. The cilia are typically arranged in preciseand species-specific patterns on the surface of the cell andare firmly embedded in a one-micrometer thick protein-rich layer that coats the outside of the cell membrane.There are about eight thousand known species of ciliates,but probably many more unidentified species. The cili-ates have little economic significance with the possibleexception of Balantidium coli, a species that inhabits thehuman gut and causes a rare form of dysentery. Stentorand Blepharisma belong to a group of ciliates called thepolyhymenophorans, all of which have complex ciliarystructures.10 The polyhymenophoran ciliates are an ancientgroup, as fossils of these ciliates have been dated to 100million years ago.

Biologists have studied light-induced responses of theciliates for over 100 years.11 Sixteen species of ciliates ex-hibit some form of phototaxis, moving toward the light(positive phototaxis), away from the light (negative pho-totaxis), or perpendicular to the light direction (transversephototaxis).12 Of these sixteen species, five—includingStentor and Blepharisma—are brightly colored due to thepresence of vesicles that contain pigment granules. Thesevesicles lie between the rows of cilia, directly beneath thecell membrane. In all five brightly colored species, the pig-ment granules presumably contain the photoreceptivepigment that controls phototaxis.

The Stentor cell is typically about 0.35 millimeters long,although some strains can grow up to 2 millimeters inlength. It assumes a pear-like shape when swimming, butis otherwise shaped like a trumpet.13 This species is namedafter the ancient Greek warrior Stentor, who, accordingto Homer, had a trumpet-like voice and “could cry out inas great a voice as fifty other men.”14 Stentor cells havelongitudinal rows of granules (0.3 to 0.7 micrometers indiameter) that are blue-green in color because they con-tain the pigment stentorin, which strongly absorbs red andUV radiation.15 Blepharisma is about the same size asStentor, but is spindle-shaped.16 Blepharisma is namedafter the Greek word for eyelid (blepharon), because its longcilia resemble eyelashes. These cells have pigment gran-ules (0.35 micrometers in diameter) that make themappear red under dim light because of the fluorescenceemitted by the pigment blepharismin.17 Blepharismaturns blue-green under strong light, however, becauseblepharismin is transformed to “blue-blepharismin”,which is only weakly fluorescent.

Behavioral Responses to LightStentor coeruleus was one of the first ciliates used to

study light-induced movement responses.18 A ring of hair-like cilia surrounds its large open end, which serves as a

Figure 3. Hypericin and stentorin.Hypericin, classified by chemists as a meso-napthodianthrone-type molecule, is a naturally occurringpigment found in Saint John’s wort and is a potentialtherapeutic agent for various diseases. Stentorin, ahypericin-like pigment, is the photoreceptive pigmentthat controls light-induced movement responses in theciliated microorganism, Stentor coeruleus; a similarpigment, blepharismin, controls movement responses inthe related species, Blepharisma japonicum. Hypericin,stentorin, and blepharismin all strongly absorb red andultraviolet radiation.

Figure 2. Blepharisma japonicum, a red-colored,photoresponsive ciliate whose size varies dependingon the strain and nutrition. Photo courtesy of FrancescoLenci, CNR Institute of Biophysics, Pisa, Italy.

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mouth and anus and is most sensitive to light and other stimuli. More sparsely distributed cilia cover the rest of itssurface. At the narrow posterior end lies the “foot”, which often attaches to stationary objects. Stentor avoids the lightand tends to collect in shaded regions by using a trial and error method—it constantly rotates about its long axis tosample the light environment, then aims toward the darkness.

Stentor and Blepharisma exhibit step-up photophobic responses: an increase in the light level causes the cilia toreverse the direction of their beating, so that the cell stops, reverses direction, and moves out of the light. These twospecies also exhibit negative phototaxis, in that they move away from a directional light source.19 When cells areirradiated with a light beam that converges to a focal point and then diverges, they swim through the bright focalpoint and away from the light source.20 This indicates that they truly sense the light direction and not merely thefluence rate (“intensity”) of the light.21 In nature, the step-up photophobic response and negative phototaxis presum-ably allow Stentor and Blepharisma cells to move toward the bottom of a pond, where they can avoid zooplanktonpredators or develop into cysts—dormant and resistant capsules.

Stentor and Blepharisma are killed when exposed to very bright light. Thus, while a one second pulse of light at 0.1W m-2, roughly equivalent to the light level during late twilight, elicits a stop response in Stentor; light of 5000 W m-2,roughly equivalent to the light level during a clear midday, kills the cells.22 Under high levels of light, stentoringenerates reactive oxygen species that react with proteins, lipids, and other molecules and this damage eventuallykills the cells. Light-induced generation of reactive oxygen species is also responsible for the phototoxic effects ofhypericin given to human patients, many of the symptoms of the metabolic disease porphyria, and the harmfuleffects of bright sunlight on plants.23

The effect of very bright light on Blepharisma is more complicated. Cells grown under dim light (which appear reddue to fluorescence from the pigment blepharismin) are killed by light of 30 W m-2 because reactive oxygen species aregenerated. If the cells are first exposed to light of about 3 W m-2, however, they are transformed into blue or colorlesscells that can survive treatment with light of 1500 W m-2 for 20 minutes or more.24

Since stentorin and blepharismin, the hypericin-like pigments of Stentor and Blepharisma, are responsible for thetoxic effects of bright light, one may reasonably ask: Why synthesize these pigments in the first place? A possibleanswer is that in nature, Stentor and Blepharisma use these hypericin-like pigments to control movement of the cellsout of dim light so they can avoid the phototoxic effect of very bright light.25

Chemistry of the Response to LightThe action spectrum for the photophobic response in Stentor coeruleus has a major peak near 610 nm, with minor

peaks near 550 nm and 480 nm.26 This is similar to the absorption spectrum of a stentorin-binding protein that hasbeen extracted from Stentor cells.27 In fact, the stentorin-binding protein is part of a large assembly of proteins thatincludes other proteins that do not bind stentorin.28 Blepharismin has been less intensively studied than stentorin.Research has shown, however, that the action spectrum for the photophobic response in Blepharisma, which has amajor peak near 590 nm and minor peaks near 540 nm and 480 nm, is similar to the absorption spectrum of ablepharismin-binding protein.29

As with all other photoreceptive pigments, the absorption of light by stentorin and blepharismin must generatechemical signals that are eventually converted into a physiological response—in this case, cell movement. While thegeneration of reactive forms of oxygen is clearly responsible for cell death under high levels of light, this reaction doesnot appear to play a role in the movement responses.30 Instead, an increase in acidity following absorption of light bythese pigments appears to be a crucial chemical reaction needed for cell movement.31 Drugs that alter the internalacidity of the cells also reduce their responsiveness to light, but have little effect on the cells’ overall viability orresponsiveness to other stimuli.32 The light-generated increase in acidity in Stentor apparently arises from the veryrapid (~10-12 second) transfer of a proton from the pigment stentorin to the associated stentorin-binding protein, andthe subsequent release of this proton to the cytoplasm.33

Recent studies suggest that the increase in acidity that follows light absorption also alters the electrical potentialacross the cell membranes of Stentor and Blepharisma. In particular, when microelectrodes are inserted into these cells,measurements show that the membranes have a resting potential of about -45 to -60 millivolts, meaning that theinside is more negative than the outside.34 About two tenths of a second after a pulse of light, the membrane under-goes a transient depolarization by about 20 millivolts, so that the difference between the inside and outside is smaller.35

Such electrochemical responses are ubiquitous among organisms. Light depolarizes the photoreceptor cells ofmany invertebrates, even though they are only distantly related to ciliates and use a rhodopsin as the photoreceptive

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pigment.36 In contrast, the rods and cones in the eyes of humans and other vertebrates hyperpolarize (become morenegative) upon illumination.

A recent hypothesis links the acidifying effect of stentorin and blepharismin, the depolarization of the membrane,and cell movement.37 According to this hypothesis, an increase in cellular acidity alters certain membrane-boundproteins that regulate the flow of ions into and out of the cell and this depolarizes the cell membrane. Membranedepolarization, in turn, alters the properties of proteins responsible for transport of calcium ions into the cell, so thatthe cells take up more calcium. The increased concentration of intracellular calcium then alters certain biochemicalreactions that control movement of the cilia. Experiments with specific inhibitors suggest that cGMP (cyclic gua-nosine monophosphate) plays an important role in transforming the light signal into a biochemical signal that con-trols cilia movement.38 This same compound is also important in controlling the vision of vertebrates and inverte-brates.39

Some people may think that study of the light-induced movement responses of insignificant microorganisms likeStentor and Blepharisma is an arcane pursuit with little significance to problems in the real world. However, the photo-receptive pigments in these organisms resemble hypericin, which has shown promise as an anti-viral agent. More-over, hypericin is also present in Saint John’s wort, which appears to be an effective anti-depressant. In addition, thechemical pathways that Stentor and Blepharisma use to convert the light signals received by their pigments into physi-ological movement responses share many features with the physiological pathways used for vision, hormone signal-ing, and other responses in humans.

AcknowledgementsI am grateful to Professor Pill Soon Song , University of Nebraska, Lincoln, for his encouragement and comments

on a previous version of this article.

References1. Meruelo, D. et al. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 5230-5234; Lavie, G. et al. Proc. Natl. Acad. Sci. U.S.A.

1989, 86, 5963-5967.2. Equine infections anemia virus: Carpenter, S.; Kraus, G. A. Photochem. Photobiol. 1991, 53, 169-174; human

immunodeficiency virus: Degar, S. et al. AIDS Res. Hum. Retroviruses 1992, 8, 1929-1936.3. Gulick, R. M. et al. AIDS Clinical Trials Group Protocols 150 and 258. Ann. Intern. Med. 1999, 130, 510-514;

Lenard, J. et al. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 158-162; Cooper, W.; James, J. Sixth International Conferenceon AIDS, 1990, 6(2), 369; Lavie, G. et al. Transfusion 1995, 35, 392-400; Steinbeck-Klose, A.; Wernet, P. NinthInternational Conference on AIDS, 1993, 9(1), 470. Recently, Piscitelli et al. showed that St. John’s wort interfereswith the action of indinavir, a HIV-1 protease inhibitor. Piscitelli, R. C. et al. Lancet 2000, 355, 547-548.

4. Miller, A. L. Alternative Medicine Review 1998, 3, 18-26; Linde, K. et al. Br. Med. J. 1996, 313, 253-258; Philipp, M.et al. Br. Med. J. 1999, 319, 1534-1538. A recent study indicates that St. John’s wort is ineffective in treating majordepression. Shelton, R. C. et al. J. Am. Med. Assoc. 2001, 285, 1978-1986.

5. Bennett, D. A. et al. Annals of Pharmacotherapy 1998, 32, 1201-1208.6. Cott, J. M. Pharmacopsychiatry 1997, 30 (suppl. 2), 108-112; Baureithel, K. H. et al. Pharm. Acta Helv. 1997, 72,

153-157.7. Barondes, S. H. Molecules and Mental Illness; Freeman: New York, 1993.8. DeSmet, P. A.; Nolen, W. A. Br. Med. J. 1996, 313, 241-242. For example, a recent study (Harrer, G. et al.

Arzneim.-Forsch. 1999, 49, 289-296) showed that a St. John’s wort extract (LoHyp-57, 800 mg per day) was aseffective as low dose Prozac (20 mg per day) in elderly patients with mild or moderate depression.

9. Thomas, C.; Pardini, R. S. Photochem. Photobiol. 1992, 55, 831-837; Diwu, Z. Photochem. Photobiol. 1995, 61,529-539.

10. Margulis, L. et al. Five Kingdoms: An Illustrated Guide to the Phyla of Life on Earth; Freeman: New York, 1998.There are numerous online images of Stentor and Blepharisma including <mac2031.fujimi.hosei.ac.jp/PDB/Images/Ciliophora/Stentor/>, which shows various species in the genus and a digital video gallery at <http://micro.magnet.fsu.edu/moviegallery/pondscum/protozoa/stentor/>.

11. Jennings, H. S. Behavior of the Lower Organisms; Columbia University Press: New York, 1906; Mast, S. O. Lightand the Behavior of Organisms; Wiley: New York, 1911.

12. Kuhlmann, H.-W. Naturwissenschaften 1998, 85, 143-154.

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13. Tartar, V. Biology of Stentor; Pergamon: New York, 1961.14. Lattimore, R. (translator) The Illiad of Homer; University of Chicago Press: Chicago, 1971.15. Huang and Pitelka. J. Cell Biol. 1973, 57, 704-728.16. Giese, A. C. Blepharisma: The Biology of a Light-Sensitive Protozoan; Stanford University Press: Stanford, 1973.17. Checcucci, G. et al. J. Am. Chem. Soc. 1997, 119, 5762-5763.18. Jennings, 1906; Mast, 1911.19. A related species, Stentor niger, exhibits positive phototaxis, movement toward a light source (Tuffrau, M.

Bulletin Societe Zoologie de France 1957, 82, 354-356).20. Song, P. S. et al. Photoreception and photomovement in Stentor coerulus. In Biophysics of Photoreceptors and

Photomovements in Microorganisms; Lenci, F. et al., Eds.; Plenum: New York, 1991; pp 267-279.21. Machemer, H.; Teunis, P. F. M. Sensory-motor coupling and motor responses. In Ciliates – Cells as Organisms;

Hausmann, K., Bradbury, P. C., Eds.; Lubrecht and Cramer: Amsterdam, 1996; pp 379-402.22. Ghetti, F. et al. J. Photochem. Photobiol., B 1992, 15, 185-198.23. Gulick et al., 1999.24. Giese, 1973.25. Giese, A. C. The photobiology of Blepharisma. In Photochemical and Photobiological Reviews; Smith, K. C., Ed.;

Plenum: New York, 1981; pp 139-180.26. Song. P. S. et al. The photoreceptor in Stentor coerulus. In The Biology of Photoreception; Cosens, D. J., Vince-Prue,

D., Eds.; Cambridge University Press: New York, 1983; pp 503-520.27. Dai, R. et al. Biochim. Biophys. Acta 1995, 1231, 58-68.28. Song, P. S. Journal of Photoscience 1995, 2, 31-35.29. Gioffre, D. et al. Photochem. Photobiol. 1993, 58, 275-279; Checcucci, G. et al. J. Am. Chem. Soc. 1997, 119,

5762-5763. For peaks, see: Scevoli, P. et al. J. Photochem. Photobiol., B 1987, 1, 75-84.30. Song, 1995.31. Wood, D. Photochem. Photobiol. 1976, 24, 261-266; Wells, T. A. et al. J. Phys. Chem. 1997, 101, 366-372; Fabczak, H.

et al. J. Photochem. Photobiol., B 1993a, 21, 47-52; Matsuoka, T. et al. Photochem. Photobiol. 1992, 56, 399-402.32. Fabczak et al., 1993a; Fabczak, S. et al. Photochem. Photobiol. 1993b, 57, 696-701; Fabczak, S. et al. Photochem.

Photobiol. 1993c, 57, 872-876.33. Dai et al., 1995.34. Fabczak et al., 1993b; Fabczak et al., 1993c.35. Wood, 1976.36. Fein, A.; Szuts, E. Z. Photoreceptors: Their Role in Vision; Cambridge University Press: New York, 1982. In

contrast, vertebrate rod and cone cells hyperpolarize (become more negative) upon illumination.37. Song, 1995. An alternative hypothesis suggests that electron transfer from the hypericin-like pigments plays an

important role (Wells et al., 1997).38. Fabczak, S. et al. Photochem. Photobiol. 1993d, 57, 702-706; Fabczak, H. et al. Photochem. Photobiol. 1993e, 57,

889-892.39. B. Rayer et al., 1990, Phototransduction: Different mechanisms in vertebrates and invertebrates, J. Photochem.

Photobiol. B 7, 107-148.

About the AuthorPeter A. Ensminger is a biomedical consultant and writer. This article is adapted from a chapter in his recent book,

Life Under the Sun (2001, Yale University Press), a collection of photobiology essays. Ensminger graduated withhonors from Rutgers University and earned a doctorate in biology from the University of Michigan. He was a post-doctoral fellow at Syracuse University and Cornell University and an Alexander von Humboldt Fellow at FreiburgUniversity in Germany. More information about Life Under the Sun is available from the Yale University Press website, <www.yale.edu/yup/lifesun> or the author’s own web site, <home.twcny.rr.com/geomanagement/ensmingr/lifesun.html>. Ensminger can be reached at <ensmingr@twcny.rr.com>.

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