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IntroductionThe last two centuries have witnessed the growth of organic photochemistry from a relatively

unknown to a developed discipline. During this period, photochemists have discovered new reac-tions, established mechanisms of photoreactions, laid out the ground rules for the behavior of mol-ecules in the excited state surfaces, and found applications of photochemistry in everyday life. Inspite of these achievements, photochemistry is yet to become a sought after tool in industrial syn-thetic processes. With the current emphasis on “green chemistry”, the place for photochemistry inindustrial synthetic processes seems assured. The discovery of photodynamic therapy and keratec-tomy has just brought to light the tremendous potential of “hυ” in medical technology. Light as atool can reach almost all internal organs of a body and thus its role as a “surgeon’s tool” appears areal possibility. The importance of photochemistry in materials science has long been recognizedthrough the discoveries of photography, lithography, and xerography. The future role of photo-chemistry in materials science is well placed through recent interests in non-linear optics and mo-lecular sensors, switches, and triggers. Realization of solar energy as an environmentally safe en-ergy source is yet to be fully realized. Given these potentials, activities in photochemistry are ex-pected to continue for many decades in the next millennium.

Organic photochemistry grew out of the important observations made by a number of Europeanphotochemists, Sestini, Cannizarao, Liberman, Klinger, Ciamician, Silber, Paterno, and others dur-ing the last and early part of this century.1 After a gap of almost 50 years, activities in organic photo-chemistry were initiated in the laboratories of Barton, Hammond, Zimmerman, Srinivasan, Yang,and others. Studies in these laboratories paved the path for others to pursue. Research activities inthe laboratories of Lewis, Norrish, Porter, and Kasha provided the needed information onphotophysics of organic molecules. Organic photochemistry seemed to have developed in threestages: (a) discovery of a reaction; (b) mechanistic pursuit; and (c) gaining control on the outcome ofa reaction. This article is concerned with the third aspect of the development of a photoreaction.2

As a human being we like to exercise “control” on things around us. We often achieve this goalthrough effective use of “space”. For example, when children misbehave at home they are often sentto their room. Generally, unruly citizens are restricted to small cells. Can one extend the above naïveconcept to molecules, i.e., can the molecular behavior be controlled through confinement? The smallspace we have been exploring in this context is the interior of zeolites.3 We show below withselected examples that one can achieve a certain amount of control on enantio- and regio-chemistry of products obtained in a reaction through the effective use of zeolite as areaction medium.

ZeolitesZeolites are crystalline aluminosilicates with open framework structures.3 The primary building

blocks of these materials are [SiO4]4- and [AlO4]

5- tetrahedra. These tetrahedra are linked by all theircorners to form channels and cages of discrete size with no two aluminum atoms sharing the sameoxygen. The zeolites used in most of our studies are X and Y. The cage structure of these zeolites are

Continued on page 3

Enforcing Molecules To Behave

Abraham Joy, Manoj V. Warrier and V. RamamurthyDepartment of Chemistry, Tulane University

The Spectrum Page 2

From the Executive Director

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

In This Issue

Enforcing Molecules to Behave ........................................................................................................................................... 1From the Executive Director ................................................................................................................................................. 2Resonance Energy Transfer as an Emerging Strategy for Monitoring Protein-Protein InteractionsIn Vivo: BRET vs. FRET......................................................................................................................................................... 9Luminescent Exciplexes and Excimers Resulting From Metal-Metal Bond Formation .............................................. 15

This fall, First Solar LLC constructed a facility in an industrial park near Perrysburg, Ohio, just a few miles north ofBowling Green. First Solar is one of the leading companies in the world to step into the photovoltaic solar energyconversion fray commercially. First Solar will manufacture flat glass panel laminates for photovoltaic energy conver-sion. Each panel is comprised of 116, 1–cm wide, series-connected Cd/Te cells, and produces a rated operating powerof approximately 50 watts.

First Solar is the latest edification of Harold McMaster’s ingenuity and tenacity. At least 15 years ago, Haroldbegan talking to me about photovoltaics. Specifically, he felt that he and his colleagues could manufacture flat glasspanels comprised of arrays of solar cells targeted at utility applications. Since this time, Dr. McMaster has kept me upto date with the latest in opportunities for his technology as various power companies, and even countries, put costtargets for “clean power generation” on the table.

Getting First Solar to this stage has been an extraordinary accomplishment. It was started with the help of inves-tors from the Toledo area. When promise remained promise not return, the investors grew increasingly impatient.Could a man of Harold’s reputation, and track record, have bitten off more than he could chew?

But Harold stayed the course against enormous odds. His conviction was that if he could find the right photovol-taic materials, he knew enough about glass to make the idea work. Finding the right photovoltaic was just one of theproblems he had to solve, and really not the hardest one. Connecting a plethora of small cells in an array, protectingthem from the environment and ensuring the panels would last for 20 or 30 years depending on the specification in allsorts of weather, and a host of other problems were on Harold’s mind all the time.

Making solar energy conversion commercial took more than tenacity. It took about 50 million dollars worth ofinvestment at current count. It took people. Harold’s solar enterprises maintained a staff of nearly 40 scientists andengineers for years. And it took dedication to an idea. More than once, and even though he was approaching 80 yearsof age, Harold thrust himself into the arena to retarget the enterprise toward his objective.

At this stage, Harold McMaster’s edification of photovoltaic solar energy conversion is nearly launched as a com-mercial company. And Harold’s off to his next project. I’m sure every photoscientist in the world wishes First Solarthe very, very best.

Page 3 The Spectrum

Continued from page 1

constructed of openings containingfour- and six-membered rings of[SiO4]

4- and [AlO4]5- polyhedra called

sodalite cages which surround a largesupercage (Figure 1).

Due to the difference in charge be-tween the [SiO4]

4- and [AlO4]5- tetra-

hedra, the total charge of an alumi-num containing zeolite framework isnegative and hence must be balancedby a cation, typically an alkali or al-kaline earth metal cation. Moleculesadsorbed into the zeolites are locatedin the cavities of the zeolites and ac-cess to these cavities is through a poreor window, which can be of the samesize or smaller than the size of the

cavities. It is this pore dimension which determines the size of the molecule that can be adsorbed into these structures.Depending on the pore dimensions (3-8 Å), there are small pore zeolites, medium pore zeolites and large pore zeo-lites. Zeolites X and Y are large pore zeolites and have the following unit cell composition:

X type M86(AlO2)86(SiO2)106.264 H2OY type M56(AlO2)56(SiO2)136.253 H2O

where M is the charge compensating monovalent cation. These cations occupy three different positions within thezeolites X and Y (Figure 1). The free volume available for the adsorbed organic molecule within the supercage de-pends on the number and nature of the cations. The cations and associated water molecules can significantly alter thenature of interaction between the host and the guest.

Enantioselectivity in PhotoreactionsA multitude of elegant and extremely efficient chiral induction strategies have been designed for numerous ther-

mal reactions during the last decade. There are a considerably less number of examples of photochemical asymmetricinductions.4 A recent successful approach in this context has been to make use of a confined media such as inclusioncomplexes and crystals.5 While crystalline and host–guest assemblies have been useful to conduct enantioselectivephotoreactions, their general applicability has been limited. We believe that zeolites offer a solution to this limitation.Zeolites, when functionalized with chiral inductors, can be more general with respect to the types of molecules thatcan be made to undergo enantioselective photoreactions. Zeolites can include a large number of different types ofmolecules, with the only limitation being that the dimensions of the guest must be less than the pore dimensions ofthe zeolite. In spite of the tremendous potential, currently chiral zeolites are not available. Due to the absence ofreadily available chiral zeolites, our approach is to adsorb chiral organic molecules within the cages of a zeolite. Overthe past few years we have examined a number of systems with the hope of understanding the principles whichgovern asymmetric photoreactions within zeolites. These have included the electrocyclization of tropolone ethers, theγ-hydrogen abstraction of adamantyl ketones and cyclohexyl ketones, the oxidation of olefins and the di-π methanerearrangement of benzonorbornadiene.6

Upon excitation, tropolone alkyl ethers undergo 4πelectron disrotatory ring closure to yield racemic prod-ucts; depending on the mode of disrotation oppositeenantiomers are obtained (Scheme 1). Enantiomericallypure products can be obtained by controlling the modeof ring closure. One obvious approach is to adsorb

Figure 1. The structure of a supercage in X and Y zeolites. Cation locations areshown as dark circles and marked as Type I, II and III. The size of the supercageis indicated in the left part of the figure with a comparison to that of pyrene.

10.0Å

Pyrene

Covalent~1.5Å

Ionic~2.8Å

7.8Å9.1Å

Type III (X)

Type I (X)

Type II (X,Y)

Type III (X)

Type I (X)

Type II (X,Y)

and (Y)

Scheme 1

O

H3C

O

OCH3

OOCH3

Ohυ'In'

'Out'

hυ

The Spectrum Page 4

tropolone alkyl ethers on a sur-face that is likely to restrict oneof the two modes of rotation.For example tropolone alkylether is expected to experiencedifferent extents of steric inter-action with the surface when itrotates inwards or outwards(Figure 2).

Since tropolone alkyl ether isnot expected to show a prefer-ence for adsorption from oneenantiotopic face over the other(Figure 2), adsorption on a sur-face by itself is not expected toinfluence enantioselectivity. Toachieve chiral induction prefer-ential adsorption from oneenatiotopic face of the tropolone

alkyl ether may be brought about by chirally modifying the surface. In Figure 2 a cartoon representation of how achiral inductor present on a surface may control the mode of adsorption by tropolone alkyl ether is provided. Asurface that can hold the chiral inductor firmly in place is required to achieve the desired goal. Surfaces such as silicaand alumina, which do not contain cations, are expected to be less effective in this regard.

Cations in zeolites are expected to strongly interact with chiral inductors and thus present them in certain geom-etries to the reactant molecule. Based on this rationale we examined the photochemistry of tropolone alkyl etherswithin chirally modified zeolites. The enantiomeric excesses (e.e.) obtained with tropolone alkyl ethers are dependent

on the chiral agent, the alkoxysubstituent, the water contentwithin the supercage, the natureand number of cations, and thetemperature. The results ob-tained with tropolone alkylethers clearly illustrate thecomplexities of chiral inductionwithin a zeolite and point outthe parameters that need tobe optimized to obtain high e.e.The high e.e. (69%) obtainedwith tropolone ethyl phenylether is most encouraging (seeFigure 3).

Based on the observationthat the best e.e. is obtainedwith bifunctional chiral agents(ephedrine, pseudoephedrine,norephedrine, and valinol), wetentatively conclude that amulti point interaction betweenthe reactant molecule, the chiralinductor, and the zeolite interioris necessary to induce preferen-tial adsorption of tropolone

Figure 2. Adsorption of tropolone alkyl ether (TAE) on a surface: Chiral inductor may

control the enantiotopic face by which TAE adsorbs. In the absence of a chiralinductor TAE will show no preference for adsorption from either enantiotopic face.Note that the same chiral inductor interacts differently when the TAE adsorbs through

different enantiotopic faces.

Figure 3: HPLC traces of product distribution from irradiation of tropolone ethylphenylether within ephedrine modified NaY zeolite. Note NaY gives 0 e.e. and ephedrine

gives ~69% and the isomer that is enhanced is reversed by the antipodes of ephedrine.

OO O O

O O

HO

NHCH3

CH3

NaY

Ephedrine

O O

OO

NaY NaY / (-)-Ephedrine NaY / (+)-Ephedrine

e.e. 0% e.e. 68% e.e. 69%

Page 5 The Spectrum

alkyl ether from a single enantiotopic face.The recognition points in these cases are mostlikely the hydroxyl, amino, and aryl groupsof the inductor, the cations of the zeoliteand the carbonyl and methoxy groups oftropolone alkyl ether. The dependence ofchiral induction (% e.e.) on the nature andnumber of cations suggests a crucial role forthe cation present in the supercages in thechiral induction process. This is furtherstrengthened by the results observed with wetand dry zeolites. The presence of water de-creases chiral selectivity. Results in one caseare shown in Figure 4. Water molecules thatare expected to hydrate the cation willmake the latter less effective in holding thetropolone alkyl ether on the zeolite surface.They also disrupt the close interaction be-tween the reactant and the chiral inductor. Asimple cartoon representation for the influ-ence of water is presented in Figure 4.

At present we do not have enough infor-mation to build a comprehensive model thatwould be applicable to a variety of systems.Based on the preliminary data, we concludethat our ability to control the geometry ofadsorption of a reactant within a cavity of azeolite will play an important role in achiev-

ing high e.e. during a photochemical reaction. Therefore the choice of the chiral inductor and the conditions of thereaction may prove to be the deciding factors in achieving high enantioselectivity.

Site (Regio) Selectivity in PhotoreactionsPhoto-Fries rearrangement of phenyl ac-

etate and photo-Claisen rearrangement ofallylphenyl ether yield ortho-hydroxy andpara-hydroxy isomers as products (Schemes2 and 3). In solution, independent of the po-larity of the medium, one obtains a mixture.On the other hand, zeolite once again comeshandy to control the product distribution.7

The best visual example of the influence ofzeolite on product distribution during aphoto-Fries reaction can be found in Figure 5where the GC traces of the product distribu-tions upon photolysis of 1-naphthyl 2-methyl2-phenylpropanoate in hexane solution andwithin NaY zeolite are provided (work car-ried out in collaboration with W. Gu and R.G. Weiss).8 Remarkably, while in solutioneight products are formed, within a NaY zeo-lite a single product dominates the productmixture (Scheme 4).

O CH3

O

OH OH

CH3

O

OH

H3C

O

hυ ++

Hexane

Methanol

Na Y/hexane

20

17

5

53

44

91

27

39

4

R = CH3

O OH OH OHhυ

+ +

Hexane

Methanol

Na Y/hexane

48

16

3

21

42

79

31

42

18

Scheme 2

Scheme 3

OO

O OPhPh

Zeolite/Chiral inductor

hυ

"dry""wet"

Na Y/ (+)Ephedrine 17 (A) 69 (B)

Na Y/(–) Pseudoephedrine 8 (B) 20 (A)

Na Y/(–) Norephedrine 23 (B) 38 (A)

Enantiomeric excess (%)Chiral inductor

HO

H

HO

HO

H3CN

O O R

H H

H3C

HO

H

HOH

HOH

HO

H

HOH

HO H

HOH

OH3CN

O O R

H H

H3C

"wet""dry"

Figure 4. Dependence of e.e. on water content within chirally modifiedNaY zeolite. Note that the e.e. is enhanced within a dry zeolite and alsothe isomer that is enhanced is reversed with respect to a wet zeolite. Acartoon representation of our visualization of the cage in the presenceand absence of water molecules is also shown above.

The Spectrum Page 6

Both photo-Fries and photo-Claisen rearrangements proceed via a similar mechanism (Scheme 5). Promotion tothe excited singlet state results in fragmentation of the ester and ether. Cage escape, recombination, and hydrogenmigration result in both the ortho- and the para-isomers. However, the factors that control the outcome of the productsvary with the nature of the medium. In solution, it is the electron densities at various aromatic carbons in the phenoxyradical which control the regioselectivity. Selectivity within zeolites results from the restriction imposed on the mobil-ity of the phenoxy and the acyl fragments by the supercage and the cations. Based on a comparison of the resultsobserved in the case of phenyl acetate and allyl phenyl ether, we believe that an interaction between the cation and thetwo reactive fragments is contributing to the observed selectivity. While the size and shape of the acyl and allylradicals are expected to be similar, the strength of the interaction between the cations and these fragments will bedifferent. The weaker binding of the allyl radical is translated to an increased yield of the para-isomer in the case ofallyl phenyl ether (Schemes 2 and 3).

Recognition of the following features of the zeolite interior has helped us control site-selectivity during variousphotorearrangements: The cavity walls of zeolites, unlike those of many other organized media, are not “passive”.Cations present within zeolites help anchor the reactants, intermediates, and products to the surfaces of a reactioncavity. In addition, the walls are very “hard” so that the shapes and volumes of the cavities do not change during thetime period of reactions.

O R

O

1) R = -CH2Ph

2) R = -CH(CH3)Ph

3) R = -C(CH3)2Ph

OH

OH

R

R

O

O

OH

R

OH

R

OR OH R

R R

+ +

+

+

+

4 5 6 7

8 9 10 11

+

Scheme 4

O

O

R O

O

R

O OO

O

H

OR

OH

R

O

OH

OR

OH

RO

O

O

R

1

•OH

hυ

Cage Escape

O

O

R ••

•

O

O

R

••

•

•

•

•

•

Migration of acyl radical

Rotation of phenoxy radical

(a)

(b)Stationary

Stationary

Scheme 5

SummaryDuring the past two decades, a number of

organized assemblies (micelles, vesicles,mono and bilayers, liquid crystals, cyclo-dextrins, silica, clay and zeolite surfaces, etc.)have been examined as media to control theexcited state behavior of organic molecules.Each of them is unique in their ability tomodify photoreactions. Zeolites are far moreversatile in their ability to control reactionsof a large variety of molecules. A word of cau-tion may be appropriate when one examineszeolites as media. Depending on the cationand on the activation condition, zeolites maypossess Brønsted acid sites. Therefore, it is im-portant to characterize the zeolite being usedand neutralize the acid sites prior to employ-ing it as a medium for photoreactions.9

AcknowledgmentThe authors thank Professors J. R. Scheffer

and R. G. Weiss for their continued collabo-ration on many aspects of the work presentedin this article. The National Science Founda-tion and the Department of Energy arethanked for the financial support.

References1. Roth, H. Angew. Chem., Int. Ed. Engl. 1989, 28, 1193.2. Ramamurthy, V.; Robbins, R. J.; Thomas, K. J.; Lakshminarasimhan, P. H. In Organised Molecular Assemblies in

the Solid State; Whitsell, J. K., Ed.; John Wiley: Chichester, 1999, pp 63-140.3. Breck, D. W. Zeolite Molecular Sieves: Structure, Chemistry and Use; Wiley & Sons: New York, 1974.4. (a) Inoue, Y. Chem. Rev. 1992, 92, 741. (b) Rau, H. Chem. Rev. 1983, 83, 535. (c) Pete, J. P. Adv. Photochem. 1996,

21, 135. (d) Everitt, S. R. L.; Inoue, Y. In Molecular and Supramolecular Photochemistry, Vol. 3; Ramamurthy, V.;Schanze, K. S., Eds.; Marcell Dekker: New York, 1999; Vol. 3, pp 71. (c) Buschmann, H.; Scharf, H.D.;Hoffmann, N.; Esser, P. Angew. Chem., Int. Ed. Engl. 1991, 30, 477.

5. (a) Gamlin, J. N., Jones, R.; Leibovitch, M.; Patrick, B.; Scheffer, J. R.; Trotter, J. Acc. Chem. Res. 1996, 29, 203.(b) Leibovitch, M.; Olovsson, G.; Scheffer, J. R.; Trotter, J. Pure & Appl. Chem. 1997, 69, 815. (c) Toda, F. Acc.Chem. Res. 1995, 28, 480.

6. (a) Leibovitch, M.; Olovsson, G.; Sundarababu, G.; Ramamurthy, V.; Scheffer, J. R.; Trotter, J. J. Am. Chem. Soc.1996, 118, 1219. (b) Sundarababu, G.; Leibovitch, M.; Corbin, D. R.; Scheffer, J. R.; Ramamurthy, V. J. Chem.Soc., Chem. Commun. 1996, 2159. (c) Joy, A.; Scheffer, J. R.; Corbin, D. R.; Ramamurthy, V. J. Chem. Soc., Chem.Commun. 1998, 1379. (d) Joy, A.; Robbins, R. J.; Pitchumani, K.; Ramamurthy, V. Tetrahedron Lett., 1997, 8825.(e) Joy, A.; Corbin, D. R.; Ramamurthy, V. In Proceedings of 12th International Zeolite Conference, Traecy, M. M. J.;Marcus, B. K.; Bisher, B. E.; Higgins, J. B., Eds.; Materials Research Society: Warrendale, PA, 1999; pp 2095.(f) Joy, A.; Ramamurthy, V. Chem.—Eur. J., in press.

7. (a) Pitchumani, K.; Warrier, M.; Ramamurthy, V. J. Am. Chem. Soc. 1996, 118, 9428. (b) Pitchumani, K.; Warrier,M.; Cui, C.; Weiss, R. G.; Ramamurthy, V. Tetrahedron Lett. 1996, 37, 6251. (c) Pitchumani, K.; Warrier, M.;Ramamurthy, V. Res. Chem. Intermed. 1999, 25, 623.

8. Gu, W.; Warrier, M.; Ramamurthy, V.; Weiss, R. G. J. Am. Chem. Soc. 1999, 121, 9467.9. (a) Thomas, K. J.; Ramamurthy, V. Langmuir 1998, 14, 6687. (b) Ramamurthy, V.; Lakshminarasimhan, P.; Grey,

C. P.; Johnston, L. J., J. Chem. Soc., Chem. Commun. 1998, 2411-2424. (c) Kao, H. M.; Grey, C. P.; Pitchumani, K.;Lakshminarasimhan, P. H.; Ramamurthy, V. J. Phys. Chem. 1998, 102, 5627.

Page 7 The Spectrum

Hexane solution NaY

3

43

4

7

5

611

109Solvent Solvent

Figure 5. GC traces of the products upon photolysis of 3 in hexanesolution and within NaY zeolite. The identity of the peak is marked withcompound numbers in Scheme 4.

The Spectrum Page 8

About the AuthorsAbraham Joy and Manoj Warrier are pursuing their Ph.D.s at Tulane University. V. Ramamurthy is the Bernard-

Baus Professor of Chemistry at Tulane and can be reached by email at murthy@mailhost.tcs.tulane.edu. For moreinformation on the authors’ research activities, visit the website www.tulane.edu/~murthy/home.html.

Photophysics and Photochemistry 2000 ConferenceOctober 19-21, 2000

Oeiras, Portugal

The Photophysics and Photochemistry 2000 conference will be held at the Instituto de Tecnologia Química e Biológica(ITQB), Universidade Nova de Lisboa, Oeiras, Portugal, October 19-21, 2000, in honor of the 75th birthday ofProfessor Ralph S. Becker. The conference will be limited to 120 participants and will include five plenary lectures,twelve invited and contributed lectures, and two poster sessions. The plenary and invited lectures are given below.

Plenary Lectures Invited LecturesR. S. Becker, USA V. Balzani, ItalyM. A. El-Sayed, USA P. Barbara, USAJ. Jortner, Israel S. E. Braslavsky, GermanyJ. Michl, USA A. Horta, SpainA. H. Zewail, USA V. Bonacic-Koutecky, Germany

M. Olivucci, ItalyJ. C. Scaiano, CanadaG. B. Schuster, USAJ. Waluk, PolandU. P. Wild, SwitzerlandK. A. Zachariasse, Germany

There will be one contributed lecture selected from the submitted contributions. For details, see the world wide website at http:www.itqb.unl.pt/pp2000. For more information on the conference, contact:

Organizing Committee - PP 2000ITQB, Apartado 127P-2781-901 Oeiras, PortugalTel: (+351-21) 446 9727Tel: (+351-21) 446 9712 (Secretariat)Fax: (+351-21) 441 1277Email: pp2000@itqb.unl.pt

The Spectrum on the World-Wide Web

The Spectrum is available on the Center’s Web site: http://www.bgsu.edu/departments/photochem/. You can accessvia Acrobat Reader. There are instructions for downloading a free copy of Acrobat Reader from the Adobe Web site.

If you plan to access The Spectrum electronically, please send an e-mail to: photochemical@listproc.bgsu.edu. Wewill remove you from our paper mailing list. Please browse our Web site for up-to-date information about the Centerand its programs.

Page 9 The Spectrum

Protein-protein interactions are known to play an important role in a variety of biochemical systems. To date,thousands of protein-protein interactions have been identified by using the conventional two-hybrid system, but thismethod is limited in that the interaction must occur in the yeast nucleus. This means interactions that strictly dependupon cell-type specific processing and/or compartmentalization will not be detected. Therefore, a number of newmethods have been developed recently that rely on reconstitution of biochemical function in vivo, such as fluores-cence resonance energy transfer (FRET), protein mass spectrometry, or evanescent wave.1 Among those methods, theresonance energy transfer techniques have potential advantages for assaying protein-protein interactions in livingcells and in real time. In this article, we will describe a new resonance energy transfer method.

Fluorescence Resonance Energy Transfer (FRET)Fluorescence Resonance Energy Transfer (FRET)2,3 is a well-established phenomenon that has found limited use in

cellular microscopy. When two fluorophores (the “donor” and the “acceptor”) with overlapping emission/absorp-tion spectra are within ~50 Å of one another and their transition dipoles are appropriately oriented, the donorfluorophore is able to transfer its excited-state energy to the acceptor fluorophore. Therefore, if appropriate fluorophoresare linked to proteins that might interact with each other, the proximity of these candidate interactors could be mea-sured by determining if fluorescence resonance energy is transferred from the donor to the acceptor. Thus, the pres-ence or absence of FRET acts as a “molecular yardstick.”

The discovery and development of green fluorescent protein (GFP) and its mutants made possible their use asFRET donors and acceptors.4-10 Genetically fusing GFP derivatives to the candidate proteins enabled the detection ofprotein-protein proximity in real time in living cells of the organisms from which the proteins were originally ob-tained.7,8 In those studies, blue fluorescent protein (BFP) was used as the donor fluorophore and GFP was the accep-tor. As mentioned above, the efficiency of the resonance transfer depends upon the spectral overlap of the fluorophores,their relative orientation, as well as the distance between the donor and acceptor fluorophores. By targeting the fusionproteins to specific compartments, this FRET-based assay can also allow protein interactions to be observed withincellular compartments in vivo, as has been shown for mitochondria and nuclei.7,8 However, because FRET demandsthat the donor fluorophore be excited by illumination, the practical usefulness of FRET can be limited because of theconcomitant results of excitation: photobleaching, autofluorescence, and direct excitation of the acceptor fluorophore.Furthermore, some tissues might be easily damaged by the excitation light or might be photoresponsive (e.g., retina).

Bioluminescence Resonance Energy Transfer (BRET)Recently, we developed a bioluminescence resonance energy transfer (BRET) system for assaying protein-protein

interactions that incorporates the attractive advantages of the FRET assay while avoiding the problems associatedwith fluorescence excitation.11 In BRET, the donor fluorophore of the FRET pair is replaced by a luciferase, in whichbioluminescence from the luciferase in the presence of a substrate excites the acceptor fluorophore through the sameresonance energy transfer mechanisms as FRET.

The bioluminescent Renilla luciferase (RLUC; MW = 35 kD) was chosen as the donor luciferase in our BRET be-cause its emission spectrum is similar to the cyan mutant of Aequorea GFP (λmax ≈ 480 nm) which has been shown toexhibit FRET with the acceptor fluorophore EYFP, which is a yellow-emitting GFP mutant.6 The excitation peak ofEYFP (513 nm) does not perfectly match to the emission peak of RLUC, but the emission spectrum of RLUC is suffi-ciently broad that it provides good excitation of EYFP. The spectral overlap between RLUC and EYFP is similar to thatof EYFP and the enhanced cyan mutant of GFP, ECFP, which yields a critical Förster radius (R0) for FRET of ~50 Å.7

Thus, we would expect significant BRET between RLUC and EYFP, with an R0 for BRET of ~50 Å. The fluorescenceemission of EYFP is yellow, peaking at 527 nm, which is distinct from the RLUC emission peak. Furthermore, the

Resonance Energy Transfer as an Emerging Strategy for MonitoringProtein-Protein Interactions In Vivo: BRET vs. FRET

Yao Xu, David W. Piston, and Carl Hirschie JohnsonVanderbilt University

The Spectrum Page 10

substrate for RLUC, coelenterazine, is a hydrophobic mol-ecule that is able to permeate cell membranes, and RLUCand EYFP do not naturally interact with each other.

In the BRET assay of protein interactions, RLUC is ge-netically fused to one candidate protein, and EYFP is fusedto another protein of interest that perhaps interacts withthe first protein. If RLUC and EYFP can be brought closeenough for resonance energy transfer to occur, the biolu-minescence energy generated by RLUC can be transferredto EYFP, which then emits yellow light (Figure 1). In theBRET assay for protein interaction, this resonance transfercan occur between RLUC/EYFP fusion proteins that in-teract. If there is no interaction between the two proteinsof interest, RLUC and EYFP will be too far apart for sig-nificant transfer and only the blue-emitting spectrum ofRLUC will be detected. Thus, protein-protein interactionscan be monitored both in vivo and in vitro by detecting theemission spectrum and quantifying the emission ratio at530nm/480nm.

BRET between RLUC and EYFP was first demonstratedin control experiments in which RLUC was fused directlyto EYFP through a linkage of 11 amino acids.11 The lumi-nescence profile of the E. coli cells expressing theRLUC::EYFP fusion construct yielded a bimodal spectrum,with one peak centered at 480 nm (as for RLUC), and anew peak centered at 527 nm (as for EYFP fluorescence).11

This result suggests that a significant proportion of theRLUC energy is transferred to EYFP and emitted at thecharacteristic wavelength of EYFP. This indicates thatRLUC/EYFP could be an effective combination to applyin a protein-protein interaction assay.

Application of BRET to Clock ProteinsTo test BRET as a protein-protein assay, we chose the proteins encoded by circadian (daily) clock genes from

cyanobacteria and fused them to RLUC or EYFP, respectively. In cyanobacteria, the kaiABC gene cluster encodes threeproteins, KaiA (MW = 32.6 kD), KaiB (MW = 11.4 kD), and KaiC (MW = 58 kD) that are essential for circadian clockfunction.12 Iwasaki et al. have used the yeast two-hybrid and in vitro binding assays to discover that Kai proteinsinteract in various ways, such as formation of KaiB-KaiB homodimers.13 First, we tried the N-terminal fusions of KaiBto RLUC and to EYFP. The luminescence spectra of E. coli expressing these fusions showed a second peak in the cellsexpressing both RLUC::KaiB and EYFP::KaiB (Figure 2, right side). This spectrum is similar to that depicted for thefusion protein RLUC::EYFP.11

We further tested all possible combinations of KaiB fusions with RLUC or EYFP, including N- vs. N-, N- vs. C-,C- vs. N-, as well as C- vs. C-terminal fusions. All of these combinations of the KaiB’s fusion proteins showed BRET(unpublished data). KaiB interactions was also observed in vitro by BRET.11 To demonstrate that this bimodal spec-trum does not occur nonspecifically, we used KaiA as a control, in which EYFP was fused to a slightly truncated KaiA.The luminescence spectra of E. coli co-expressing EYFP::KaiA with RLUC::KaiB did not exhibit the second lumines-cence peak, indicating no interaction occurred between KaiA and KaiB. Our results, therefore, strongly suggest thatinteraction among KaiB molecules either in N-terminal or C-terminal fusions to the donor luciferase or the acceptorfluorophore has brought the RLUC and EYFP into close proximity such that energy transfer occurs for ~50% of theRLUC luminescence. Thus, BRET supports the data from the yeast two-hybrid assay13 demonstrating that the clockprotein KaiB self associates to form multimers.

Figure 1. A diagram of bioluminescence resonanceenergy transfer (BRET) as for a protein-protein interactionassay. One protein of interest (B) is genetically fused tothe donor luciferase RLUC, and the other candidateprotein (A) is fused to the acceptor fluorophore EYFP.Interaction between the two fusion proteins can bringRLUC and EYFP close enough for BRET to occur, withan emission of longer wavelength light.

EYFP

Light(530 nm)

Light(480 nm)

BRET

B

RLUC

Interaction

EYFP

B

RLUC

Light(480 nm)

A

A

Page 11 The Spectrum

In the experiments described above, theextent of BRET was determined by measur-ing emission spectra.11 For applications suchas microscopic imaging and high-throughputscreening, it would be more convenient tomeasure the ratio of luminescence intensitiesat two fixed wavelengths, e.g., 480 nm and530 nm. Ratio imaging has the advantage ofautomatically correcting for differences inoverall levels of expression of RLUC andEYFP fusion proteins. The top portion of Fig-ure 2 is the images of E. coli cultures express-ing fusion proteins that either exhibit (right)or do not exhibit (left) BRET. These images ofliquid E. coli cultures (5 microliter cultures)were collected using a charge-coupled device(CCD) camera through interference bandpassfilters centered at 480 nm and 530 nm, respec-tively. In the cultures co-expressing the inter-acting combination of RLUC::KaiB withEYFP::KaiB, the amount of light emitted at 480nm and 530 nm are roughly equal, as wouldbe predicted from the spectra depicted (Fig-ure 2, right). In contrast, in the cultures con-taining a non-interacting combination ofRLUC::KaiB with EYFP::KaiA, there is muchless light emitted at 530 nm than at 480 nm(Figure 2, left). As we reported previously, the

extent of BRET can be quantified according to the 530nm/480nm ratios of luminescence intensity in the image.11 Thus,the 530nm/480nm ratios can apparently be used to evaluate BRET and thereby infer if protein-protein interactionhas occurred.

New Tools for BRETVery recently, several new tools for BRET have appeared that may prove useful. The first is that there is a new

luciferase that was isolated from Gaussia that has an emission spectrum like that of RLUC but whose MW is 20 kD(available from Prolume Ltd.). Like RLUC, this luciferase uses coelenterazine as a substrate. This luciferase may haveall the advantages of RLUC, but by virtue of its smaller size, better allow native interactions without steric hinderancein fusion proteins. Another tool of potential advantage is a new fluorescent protein isolated from anthozoans.14 Theseproteins that are remote homologs of GFP form a new group of fluorescent tags. One of these proteins has a muchlonger wavelength than any other fluorescent protein yet isolated, with an excitation spectrum peaking at 558 nm anda sharp emission spectrum peaking at 583 nm. The excitation spectrum is broad enough that a luciferase like RLUCmight excite it. The advantage of this fluorescent protein is that its emission spectrum is sufficiently red-shifted thatthe separation between BRET and non-BRET luminescence is much greater than with YFP, and hence quantificationof BRET could be more accurate. This red fluorescent protein is now available from Clontech as “DsRed.”

A Potential Screening SystemBased on these data, we proposed11 a relatively simple scheme for designing an in vivo library screening system for

protein-protein interaction through BRET (Figure 3). By measuring the light emission collected through interferencefilters, the 530 nm/480 nm luminescence ratio of E. coli (or yeast) colonies expressing a “bait” protein fused to RLUCand a library of “prey” molecules fused to EYFP (or vice versa) could be measured. It would be possible to screencolonies of bacteria or yeast on agar plates using a camera imaging system. On the other hand, a photomultiplier-based instrument designed to measure luminescence of liquid cultures in 96-well plates could be adapted to

Figure 2. Comparison of complete BRET spectra using a fluorescencespectrophotometer with camera images of E.coli cells. Top, culturesimaged with a CCD-camera through filters transmitting light of 480 nmor 530 nm from the transformed E. coli strains co-expressing fusionproteins exhibiting BRET on the right side (RLUC::KaiB andEYFP::KaiB) or fusion proteins that are not exhibiting BRET on the leftside (RLUC::KaiB and EYFP::KaiA). Bottom, luminescence emissionspectra measured continously from 440 nm to 580 nm for thesame strains.

Without BRET With BRET

480nm 530nm480nm 530nm

The Spectrum Page 12

high-throughput BRET screening by insertion of switchable 480 or 530 nm filters in front of the photomultiplier tube.Colonies that show high light intensity (i.e. bright colonies) at 530 nm or exhibit an above-background ratio of the530 nm/480 nm could be selected and the “prey” DNA sequence further characterized. Thus, an efficient BRET screeningsystem could be practical by using an appropriate instrument.

Advantages of Resonance Energy Transfer Techniques (BRET and FRET)The features of BRET or FRET techniques offer some attractive advantages over other current assays for protein-

protein interactions, especially the yeast two-hybrid method, which is currently the most popular method. For in-stance, BRET or FRET methods can be applied to determine whether the interaction changes with time because themeasurement is noninvasive. BRET/FRET is suitable to assay the protein-protein interactions in different subcellularcompartments or specific organelles of the native cells, as has already been shown to work for FRET.7,8 In particular,the yeast nucleus may be a poor place for some compatible proteins to meet. This advantage of BRET/FRET could beparticularly useful in the case of interacting membrane proteins for which assays are limited with other traditionalmethods. BRET or FRET also may be used to reveal interactions that depend upon cell-type specific post-translationalmodifications that do not occur in yeast and therefore can not be assayed by the yeast-two hybrid method. By usingcell-type specific promoters and/or fusion to targeting-sequences, the GFP-based BRET or FRET indicators can beobserved specifically in the cell-type and subcellular location of choice. Moreover, BRET/FRET assays could be adoptedto monitor the dynamic processes of protein-protein interactions in vivo, such as intracellular signaling.

No Technique is PerfectAs with any technique, however, the resonance energy transfer methods have some limitations. For example, the

efficiency of both BRET and FRET is dependent on proper orientation of the donor and acceptor dipoles. Conforma-tional states of the fusion proteins may fix the dipoles into a geometry that is unfavorable for energy transfer. Further,because the fluorophore/luciferase tags are fused to ends of the potentially interacting molecules, it is possible thatsome parts of the candidate molecules are interacting without allowing the fluorophore/luciferase tags to be closeenough for energy transfer to occur. Consequently, two proteins might interact in a way that is blind to the FRET/BRET technique. In other words, a negative result with a resonance transfer technique does not prove non-interac-tion. In such a case, testing different combinations of N-terminal and C-terminal fusions in the BRET/FRET assayscould help to determine the optimal orientation in which candidate proteins interact.

The luciferase/fluorescent protein tags that are fused to the candidate interacting proteins could interfere with theinteraction by steric hinderance (this problem is true for the yeast two-hybrid assay as well). Therefore, the smallerthe tags, the less likely will be the hindrance. This is the reason why the Gaussia luciferase might prove to be superiorto RLUC. Furthermore, these luciferase/fluorescent tags might cause inactive or incorrectly folded fusion proteins.For example,the bulkiness of the GFP (and its derivatives) cylinders (20 x 30 Å) have been shown to impede correctfolding of fusion proteins.10

Another consideration in the use of GFP variants as fluorophore tags is that the slow kinetics of GFP turnover mayhamper measuring the kinetics of interaction (whereas Renilla luciferase does not suffer these same disadvantages inturnover rate). New GFPs are available that have been engineered to be less stable (ref. 15 and Clontech’s d2EGFP)and the re-engineering of BRET fluorophores to be less stable could be useful in temporal studies. Moreover, the pH-sensitivity of some of the GFPs might restrict their application to subcellular areas with a higher pH value. However,this limitation can be overcome by utilizing the mutants that are less sensitive to pH.16

BRET Versus FRETBRET has potential advantages over FRET because it does not require the use of excitation illumination. BRET

should be superior for cells that are either photo-responsive (e.g., retina or any photoreceptive tissue) or damaged bythe wavelength of light used to excite FRET. Cells that have significant auto-fluorescence would also be better as-sayed by BRET than by FRET. Moreover, while photobleaching of the fluorophores can be a serious limitation ofFRET, it is irrelevant to BRET. BRET assay requires a substrate for the luciferase. In the case of RLUC, coelenterazineis the substrate. Coelenterazine is hydrophobic and can permeate all the cell types we have tested, including bacteria(E. coli and cyanobacteria), yeast, Chlamydomonas,17 plant seedlings and calli,18 and animal cells in culture. Other workershave found that coelenterazine can permeate the cytoplasm of human 293 cells,19 the cytoplasm of primary neuronaland HeLa cells,20 the nucleus of COS7 cell cultures,21 the mitochondria of endothelial cells,22 and the ER ofarterial cells.23

Page 13 The Spectrum

In addition, FRET may be prone tocomplications due to simultaneous ex-citation of both donor and acceptorfluorophores. Specifically, even withmonochromatic laser excitation, it isimpossible with the current genera-tion of fluorescent proteins to exciteonly the donor without exciting theacceptor fluorophore to some degree.In contrast, because BRET does notinvolve optical excitation, all the lightemitted by the fluorophore must re-sult from resonance transfer. There-fore, BRET is theoretically superiorto FRET for quantifying resonancetransfer.

The major limitation that BRET suf-fers in comparison to FRET is that itsluminescence may often be too dim toaccurately measure 530/480 nm ratiosif the researcher does not have accessto a very sensitive light-measuringapparatus. Manufacturers are con-tinuously developing improved in-strumentation for measuring low-light levels, and these improvementsin technology will undoubtably aidthe further development of BRET as-says of real-time protein-protein inter-actions in living organisms.

AcknowledgmentsThis research was supported by the National Institute of Mental Health (MH 43836 and MH 01179 to CHJ) and the

National Science Foundation (MCB-9633267 to CHJ). Spectral data were acquired at the Vanderbilt Cell ImagingShared Resource, supported in part by the NIH through the Vanderbilt Cancer Center (CA68485) and the VanderbiltDiabetes Center (DK20593).

References1. Mendelsohn, A. R.; Brent, R. Science 1999, 284, 1948-1950.2. Wu, P.; Brand, L. Anal. Biochem. 1994, 218, 1-13.3. Clegg, R. M. Curr. Opin. Biotechnol. 1995, 6, 103.4. Heim, R.; Prasher, D. C.; Tsien, R. Y. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 12501-12504.5. Heim, R.; Tsien, R. Y. Curr. Biol. 1996, 6, 178-182.6. Miyawaki, A.; Llopis, J.; Heim, R.; McCaffery, J. M.; Adams, J. A.; Ikura, M.; Tsien, R. Y. Nature 1997, 388, 882-887.7. Mahajan, N. P.; Linder, K.; Berry, G.; Gordon, G. W.; Heim, R.; Herman, B. Nature Biotechnol. 1998, 16, 547-552.8. Periasamy, A.; Day, R. N. J. Biomed. Opt. 1998, 3, 1-7.9. Xu, X. et al. Nucleic Acids Res. 1998, 26, 2034-2035.

10. Gadella, T. W., Jr.; van der Krogt, G. N.; Bissseling, T. Trends Plant Sci. 1999, 4, 287-291.11. Xu, Y.; Piston, D. W.; Johnson, C. H. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 151-156.12. Ishiura, M.; Kutsuna, S.; Aoki, S.; Iwasaki, H.; Andersson, C. R.; Tanabe, A.; Golden, S. S.; Johnson, C. H.;

Kondo, T. Science 1998, 281, 1519-1523.13. Iwasaki, H.; Tanihuchi, Y.; Ishiura, M.; Kondo, T. EMBO J. 1999, 18, 1137-1145.

Figure 3. Schematic diagram of a screening system for protein-protein interactionusing ratio or visual imaging of BRET.

RLUC-fusion vector (Marker A)(N-terminal and/or C-terminal fusion)

EYFP-fusion vector (Marker B)(N-terminal and/or C-terminal fusion)

Expression cassette of Rluc::known gene

Expression of fusion protein in E. coli (Host strain for expression libraries)

Constructing cDNA or genomic DNA expression libraries in plasmid

Transformation of host strain

Expression libraries resistant to A and B (Expressing RLUC and EYFP activities)

Screening expression library by BRET imaging ratio

Imag

ing

at 4

80 n

m Im

aging

at 530 nm

Isolation of the clone containing the associated target with the protein of interest based on BRET assay

14. Matz, V. M.; Fradkov, A. F.; Labas, Y. A.; Savitsky, A. P.; Zaraisky, A. G.; Markelov, M. L.; Lukyanov, S. A, NatureBiotechnol. 1999, 17, 969-973.

15. Andersen, J. B.; Sternberg, C.; Poulsen, L. K.; Bjorn, S. P.; Givskov, M.; Molin, S. Appl. Environ. Microbiol. 1998, 64,2240-2246.

16. Miyawaki, A. et al. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 2135-2140.17. Minko, I.; Holloway, S. P.; Nikaido, S.; Odom, O. W.; Carter, M.; Johnson, C. H.; Herrin. D. L. Mol. Gen. Genet. 1999,

262, 421-425.18. Sai, J.; Johnson. C. H. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11659-11663.19. Sheu, Y. A.; Kricka, L. J.; Pritchett, D. B. Anal. Biochem. 1993, 209, 343-347.20. Brini, M.; Marsault, R.; Bastianutto, C.; Alvarez, J.; Pozzan, T.; Rizzuto, R. J. Biol. Chem. 1995, 270, 9896-9903.21. Badmington, M. N.; Kendall, J. M.; Sala-Newby, G.; Campbell, A. K. Expt. Cell Res. 1995, 216, 236-243.22. Rizzuto, R.; Simpson, A. W. M.; Brini, M.; Pozzan, T. Nature 1992, 358, 325-327.23. Rembold, C. M.; Kendall, J. M.; Campbell, A. K. Cell Calcium 1997, 21, 69-79.

About the AuthorsDr. Yao Xu received his Ph.D. in Biology at Lanzhou University, China. After a postdoctoral fellowship with Dr.

Chris Lamb at The Salk Institute, San Diego, California, he joined the faculty at Zhongshan University, China. He iscurrently a Senior Research Associate in Biology at Vanderbilt University.

Dr. David Piston received his Ph.D. in Physics from the University of Illinois in 1989. After postdoctoral research inApplied Physics at Cornell University under Prof. Watt W. Webb, he came to Vanderbilt University in 1992. He iscurrently an Associate Professor. His address is Department of Molecular Physiology and Biophysics, VanderbiltUniversity, Nashville, Tennessee 37232.

Dr. Carl Johnson received his Ph.D. from Stanford University in 1982. After a postdoctoral fellowship with Dr. J. W.Hastings at Harvard, he joined the faculty at Vanderbilt University, where he is currently a Professor. Drs. Xu andJohnson may be contacted at the Department of Biology, Box 1812-B, Vanderbilt University, Nashville,Tennessee 37235.

The Spectrum Page 14

Copyright 1999 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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F. N. Castellano, M. E. Geusz,D. C. Neckers, M. Y. Ogawa,V. V. Popik, M. A. J. Rodgers,D. L. Snavely

The Spectrum Editor: Pat GreenProduction Editor: Alita Frater

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A person may make a single copy of any or all articles in this issuefor personal use. Copying beyond that permitted by the U.S.Copyright law is allowed provided that the appropriate per copyfee is paid through the Copyright Clearance Center, Inc., 27Congress St., Salem, MA 01970. For reprint permission, please writeto the Center for Photochemical Sciences.

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

IntroductionThe formation of exciplexes and excimers in solution is a well-established phenomenon in organic chemistry.1 It is

only recently, however, that well-characterized examples involving coordination compounds have been reported.2

The majority of these species appear to result from interactions involving coordinated ligands. However, severalreports of luminescent excimer formation between square planar compounds of PtII recently have appeared.3 Unfor-tunately, the role played by Pt-Pt bonding in these cases is difficult to establish due to the possibility of π-π interac-tions between the aromatic ligands present.4

This review will focus exclusively on exciplexes and excimers formed as a result of metal-metal bonding in com-pounds where π-π ligand interactions are not possible. This effectively limits the scope of this review to aqueous,luminescent exciplexes where one partner is the lowest triplet excited state of Pt2(P2O5H2)4

4- (Pt24-; Figure 1), abbrevi-

ated here as *Pt24-, and the gas phase *Hg2 excimer and *Hg3 exciplex.

The first report of an exciplex formed as a resultof metal-metal bonding appeared twelve years ago.5

It described the exciplexes formed between *Pt24- and

T1+ ions in room temperature aqueous solutions. Al-though an analogous exciplex of *Pt2

4- with Ag+ wasobserved at the same time, initial work centered onT1+,6 and only recently have studies with Ag+ beencompleted.7,8 In the intervening years only one otherexciplex resulting from metal-metal bond formationhas been reported, the anion-anion exciplex of *Pt2

4-

with Au(CN)2- in aqueous solution.7,9 Comparisons

among these luminescent exciplexes shed light onthe factors that lead to metal-metal bonded exciplexformation between ions in aqueous solution. Boththe kinetic and thermodynamic factors appear to bewell understood, and yield some unexpected find-ings. The three aqueous exciplexes consideredall involve *Pt2

4-; therefore some of the morenotable qualities of this fascinating species will firstbe reviewed.10-12

Photochemistry and Photophysics of *Pt24-

The preparation of Pt2(P2O5H2)44- was first reported in 197713 (Max Roundhill tells an entertaining story of how a

reaction vessel containing Pt24- exploded and left a coating of intensely green luminescent residue on an overhead

fluorescent light14). Once its dimeric nature was revealed,15 its relationship to luminescent RhI-RhI dimers was imme-diately appreciated.16 Like the related RhI dimers, Pt2

4- exhibits a shortening of the metal-metal separation distanceupon absorption of light. The ground state Pt-Pt separation distance of 2.925 Å15 contracts to 2.75 Å in *Pt2

4-,17 accom-panied by an increase in both the Pt-Pt stretching force constant (0.3 N/cm to 0.9 N/cm)17,18 and reorganization en-ergy19 (13 kJ/mol to 18 kJ/mol).20 An equation21 relating M-M interaction energies to force constants and bond dis-tances yields approximate interaction energies of 50 kJ/mol and 130 kJ/mol for Pt2

4- and *Pt24-, respectively. The value

for Pt24- is similar in magnitude to that estimated for molecular AuI-AuI interactions, the strongest closed shell

interaction reported.21

Luminescent Exciplexes and Excimers Resulting FromMetal-Metal Bond Formation

Jeffrey K. NagleDepartment of Chemistry, Bowdoin College

Page 15 The Spectrum

2.92

5 Å

Pt2(P2O5H2)44–

Figure 1. Structure of Pt2(P2O4H2)44- in K4[Pt2(P2O5H2)4]

.2H2O.15

(Adapted with permission from Thiel, D. J.; Livins, P.; Stern, E. A.; Lewis,

A. Nature 1993, 362, 40-43.)

The absorption and emission spectra of aqueous Pt24- are shown in Figure 2, and some of its photophysical proper-

ties are summarized in Figure 3. A weak (Φ = 0.0008), short-lived (τ = 40 ps) blue luminescence with a band maximumat 402 nm is observed upon absorption of light at 368 nm (ε = 3.45 x 103 m2/mol, f = 0.3).10-12 This is followed by rapidand efficient intersystem crossing to the relatively long-lived (τ = 10 µs), intensely (Φ = 0.55) green (λmax = 514 nm)phosphorescent triplet state. Direct excitation into this triplet state occurs at 453 nm with much lower efficiency(ε = 12 m2/mol, f = 0.001).10-12

A simple, qualitative orbital interaction model that was developed by Gray and co-workers for RhI dimers22 wassuccessfully adapted to understand both the ground state and excited state properties of Pt2

4-.16 This simple approachpredicts a bond order of zero between the PtII atoms in Pt2

4-, which increases to one upon excitation to *Pt24-. It does

not, however, account for the attractive intramolecular (as in Pt24-) and intermolecular van der Waals interactions

between closed shell metals (“metallophilicity”).21,23 These attractive forces are largely due to electron correlation,21

and are enhanced by relativistic effects.24

Given the formation of a formal bond between the Pt atoms in *Pt24-, *Pt2

4- itself could be considered an excimer.However, it is preferable to not use this term for such species, reserving it instead for intermolecular and interatomicinteractions resulting from diffusional encounters between an excited state and the ground state of the same species.The excimers of PtII monomers containing one or more N-heterocyclic ligands can be considered as authentic excimersaccording to this definition (however, even here unbridged, outer sphere van der Waals association in the groundstate can blur such a distinction). In this vein it is noted that the term exciplex has been borrowed to describe en-hanced metal-metal interactions that occur in solids as a result of light absorption.25 While this use highlights someimportant similarities between solution and solid state behavior, it is again preferable to use the terms exciplex andexcimer for species resulting from molecular and atomic encounters in the solution and gas phases.

Exciplexes of *Pt24–

The properties of the three well-characterized exciplexes of *Pt24- are summarized in Table 16-9 (fluorescent exciplexes

and exciplexes which are formed at higher concentrations, such as *(T1+,Pt24-,T1+),6 will not be considered here26). In

Table 1, σmax, τ, and Φ all refer to the phosphorescence exhibited by the three exciplexes; K, ∆Go, ∆Ho, and ∆So refer tothe exciplex formation reactions between *Pt2

4-and the three ions.

The Spectrum Page 16

Figure 3. Frontier orbital diagram illustrating some of thephotophysical properties of Pt2(P2O4H2)4

4–. All values givenare for deoxygenated aqueous solution at roomtemperature.10-12

Figure 2. Absorption and emission spectra of 1 x 10–5-mol/LPt2(P2O4H2)4

4– in aqueous solution at room temperature(1 m2/mol = 10 L/mol cm).

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.8 2.0 2.2 2.4 2.6 2.8 3.0

Abs

orba

nce

Rel

ativ

e L

umin

esce

nce

Inte

nsit

y

phos

phor

esce

nce,

Φ =

0.5

5

Wavenumber, µm–1

x100 x100 spin

-allo

wed

abs

orpt

ion,

ε

= 3

.45

x 10

3 m2 /m

ol

spin

-for

bidd

en a

bsor

ptio

n, ε

=

12 m

2 /mol

fluo

resc

ence

, Φ =

0.0

008

1.95

2.212.49

2.72

Wavelength, nm

556 500 455 417 385 357 333

fluorescenceσmax = 2.49 µm–1

τ = 40 ps

phosphorescenceσmax = 1.95 µm–1

τ = 10 µsLUMO

HOMO

intersystem crossing

spin-allowedabsorption σmax = 2.72 µm–1

f = 0.3

spin-forbidden absorptionσmax = 2.21 µm–1

f = 0.001

Table 1. Photophysical Properties of *Pt24– Exciplexesa

Exciplex σmax, µm-1 τ, µs Φ K, L/mol ∆Go, kJ/mol ∆Ho, kJ/mol ∆So, J/mol K*(Pt2

4-,Tl+) 1.79 10.0 0.55 2 x 105 –30 –19 +36*(Pt2

4-,Ag+) 1.80 2.9 0.48 1 x 105 –28 +6 +114*(Pt2

4-,Au(CN)2-) 1.74 2.2 0.030 4 x 102 –15 –49 –115

aAll values are for aqueous solutions at 25 °C.

Values for all rate constants in the proposed diffusion-based mechanism for exciplex formation6 have been deter-mined for all three exciplexes, and are reported and discussed elsewhere.6-9 The ability to characterize these exciplexesstems from the intense luminescence exhibited for all three species. Although these new luminescence bands alloverlap with the 1.95-µm-1 band of *Pt2

4-, they are at lower enough energies such that they appear as distinct shoul-ders. Therefore their integrated intensities can be determined through a Gaussian curve-fitting analysis of the ob-served luminescence lineshapes.

A simple explanation for the enhanced tendency to form intermolecular *M-M bonds compared to M-M bonds isfound in the frontier orbital diagram shown in Figure 4. It is readily apparent that while the ground state interactionleads to a net bond order of zero between the metals, when one of the metals is in an electronically excited state theresultant bond order becomes one. This simple analysis assumes the HOMO and LUMO energies of the two speciesinvolved in forming an exciplex are equal, an assumption that is not exact even in the case of excimer formation. Italso assumes a significant degree of frontier orbital localization on the metal atom(s) in the excited species.

Long-lived exciplexes are not expected to form if efficient excited state quenching occurs via either electron trans-fer or energy transfer upon diffusional encounters of the excited state and ground state species. Thus, unlike T1+,isoelectronic aqueous Pb2+ ions efficiently quench the luminescence of *Pt2

4- by oxidation to give (presumably) Pb+ andPt2

3-, with no evidence of exciplex formation.27 The Tl+/T10 standard electrode potential (–1.94 V;28 this and all othervalues below are for aqueous solutions versus SHE) is large enough compared to that for Pt2

3-*/Pt24- (1.6 V29) to pre-

clude oxidative quenching by T1+ to give Pt23- and T10. In contrast, the relatively low value for Pb2+/Pb+ (-1.0 V30) is

consistent with oxidative quenching of *Pt24- by Pb2+. As with T1+, the Au(CN)2

-/Au(CN)22- and Ag+/Ag0 potentials

(<–1.6 V31 and –1.74 V,32 respectively) also make oxidative quenching energetically unfavorable. Given values of 1.0V10 for the *Pt2

4-/Pt25- redox couple and 245 kJ/mol10 for the *Pt2

4- triplet energy, both reductive excited state electrontransfer and energy transfer quenching of *Pt2

4- by all three species are also energetically unfeasible (E0 values forT12+/T1+, Ag 2+/Ag+, and Au(CN)2

0/Au(CN)2- are 2.22 V,33 1.98 V,34 and ≈2.2 V;35 the triplet energies of T1+, Ag+, and

Au(CN)2- are all greater than 330 kJ/mol. More extensive accounts of all of these competing excited state reactions of

*Pt24- are available elsewhere.10-12

When such competing excited state deactivation path-ways are not available, it is logical to ask what factors de-termine whether exciplex formation is likely to occur be-tween ions in aqueous solution. The simple expectation thatexciplex formation between oppositely charged ions wouldbe favored over like-charged ions is at first glance confirmed.Thus, the equilibrium constants for the formation of*(Pt2

4-,Tl+) and *(Pt24-,Ag+) are both substantially greater than

that for *(Pt24-,Au(CN)2

-). A hint that the situation may bemore complicated is evident from the spectroscopic data.Specifically, consider the shifts in the luminescence maximabetween the band for *Pt2

4- and those for the three exciplexes(Table 1). The largest shift is for *(Pt2

4-,Au(CN)2-), the exciplex

with the smallest equilibrium constant of formation. Tem-perature dependence studies of all three exciplexes7-9 pro-vide estimates of the thermodynamic parameters forexciplex formation (Table 1; the estimated uncertainties inthe thermodynamic values are all less than 15%.

Page 17 The Spectrum

Figure 4. Frontier orbital diagram showing the enhancedattraction of the metal atoms for each other in a metal-metal bonded exciplex (*M-M) compared to thecorresponding ground state van der Waals encountercomplex (M-M).

M MM M *M *MM MNet M-M Bond Order = 0 Net M-M Bond Order = 1

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It is readily apparent that entropy factors play a large role in exciplex formation under these conditions. What ismost striking about the thermodynamic data is the difference in standard entropies of formation between *(Pt2

4-,Ag+)and *(Pt2

4-,Au(CN)2-). The large negative value for *(Pt2

4-,Au(CN)2-) is typical of entropies for exciplex formation in

solution, whereas the large positive value for *(Pt24-,Ag+) is not. It has been proposed that this represents a difference

in solvation effects.8,9 In the case of Au(CN)2-) the strongest anion-solvent interactions presumably occur at the cyano

ligands, the nearly neutral Au atom interacting only weakly with the water molecules. Therefore, little desolvationshould accompany exciplex formation with *Pt2

4-, and the entropy change is dominated by the formation of the Pt-Aubond. In contrast, Ag+ is relatively strongly solvated, and significant desolvation is proposed to accompany exciplexformation with *Pt2

4-. Such solvation effects account also for the large difference in enthalpy values for these twoexciplexes—the formation of *(Pt2

4-,Ag+) is even slightly endothermic. These results clearly illustrate that Coulombicion-ion interactions can play a secondary role to desolvation effects in exciplex formation between ions in polarsolvents. This is especially so for the formation of *(Pt2

4-,Au(CN)2-), the first anion-anion exciplex ever reported.7-9

The *Hg2 Excimer and *Hg3 ExciplexThe gas phase Hg atom provides a simple model for Pt2

4- since it is a closed-shell species with a 6p lowest unoccu-pied orbital capable of overlapping with other metal atoms. Like *Pt2

4- in solution,10-12 *Hg in the gas phase is lumines-cent and undergoes analogous H-atom abstraction reactions with organic molecules.36 In a similar vein, since Hg isisoelectronic with T1+, the *Hg2 excimer37 can in some ways serve as a simple gas-phase model for the metal-metalbonded exciplexes of *Pt2

4-. *Hg2 exhibits luminescence at 335 nm and has been proposed as a candidate for an excimerlaser.38 Like *Pt2

4-, *Hg2 exhibits a shorter M-M bond distance (2.604 ± 0.005 Å)39 and greater force constant(1.23 N/cm)40 than in its electronic ground state, the van der Waals molecule Hg2 (bond distance: 3.63 ± 0.04 Å; forceconstant: 0.0202 N/cm).41 The linear *Hg3 exciplex luminesces at 485 nm40 and is perhaps an even better model for the*Pt2

4- exciplexes. This is because it is formed from the gas phase reaction between *Hg2 and Hg, analogous to thesolution phase formation of *(Pt2

4-,T1+) from *Pt24- and T1+.

ConclusionAdditional examples of exciplexes and excimers formed as a result of metal-metal bonding are likely to be found.

Coordinatively unsaturated d8 square planar and d10 linear compounds can be considered isolobal with gas phase orweakly solvated s2 ions and atoms. Therefore, various combinations of these isolobal species can be expected to formluminescent exciplexes under the right conditions, in particular with favorable solvation effects. Given the relativeenergies of the lowest excited states of these species, it is expected that more examples of square planar PtII monomerand dimer (or even oligomer) excited states will be found to form exciplexes and excimers with other members of thisisolobal family. Similarly, excited state AuI compounds can be expected to readily form metal-metal bonded exciplexesand excimers with ground state AuI compounds and s2 ions and atoms. Interestingly, luminescent exciplexes of anexcited state AuI dimer with both solvent molecules and anions recently have been reported,42 providing additionalexamples of the remarkable ability of exposed metal atoms in electronically excited states to undergo a rich array ofphotochemical reactions.

References1. Birks, J. B. Photophysics of Aromatic Molecules; Wiley-Interscience: New York, 1970; Ch. 7, 9. Gilbert, A.; Baggott,

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8. Jochnowitz, E. B.; Jefferis, C. A.; Chuong, B.; Segovis, C. S.; Doede, T. M.; Hinkle, R. J.; Nagle, J. K. manuscriptin preparation.

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and references therein.

About the AuthorDr. Nagle is a professor of chemistry at Bowdoin College. A 1975 graduate of Earlham College, he received his

Ph.D. from the University of North Carolina at Chapel Hill in 1979. His research interests include inorganic photo-chemistry and photophysics, metal-metal bonding, and electron transfer. His address is Department of Chemistry,6600 College Station, Bowdoin College, Brunswich, Maine 04011-8466, email: jnagle@bowdoin.edu.

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