Photochemistry and Photobiology, 2014, 90: 419–430

Photophysics of Glycosylated Derivatives of a Chlorin, Isobacteriochlorinand Bacteriochlorin for Photodynamic Theragnostics: Discovery of aTwo-photon-absorbing Photosensitizer†

Amit Aggarwal#1,2, Sebastian Thompson#1, Sunaina Singh1,3, Brandon Newton4, Akeem Moore5, RuomieGao4,6, Xinbin Gu5, Sushmita Mukherjee7 and Charles Michael Drain*1,81Department of Chemistry, Hunter College of the City University of New York, New York, NY2Department of Science, Borough of Manhattan Community College of the City University of New York, New York, NY3Department of Natural Sciences, LaGuardia Community College of the City University of New York, New York, NY4Department of Chemistry and Biochemistry, Jackson State University, Jackson, MS5Howard University, Washington, D.C.6Department of Chemistry and Physics, State University of New York at Old Westbury, Old Westbury, NY7Department of Biochemistry, Weill Cornell Medical College, New York, NY8The Rockefeller University, New York, NYReceived 23 August 2013, revised 16 September 2013, accepted 18 September 2013, DOI: 10.1111/php.12179

ABSTRACT

The photophysical properties of a chlorin, isobacteriochlorinand bacteriochlorin built on a core tetrapentafluorophenyl-porphyrin (TPPF20) and the nonhydrolyzable para thiogly-cosylated conjugates of these chromophores are presented. Thephotophysical characterization of these compounds was donein three different solvents to correlate with different environ-ments in cells and tissues. Compared with TPPF20 other dyeshave greater absorption in the red region of the visible spec-trum and greater fluorescence quantum yields. The excitedstate lifetimes are from 3 to 11 ns. The radiative and nonradia-tive rate constants for deactivation of the excited state wereestimated from the fluorescence quantum yield and excitedstate lifetime. The data indicate that the bacteriochlorin hasstrong absorption bands near 730 nm and efficiently enters thetriplet manifold. The isobacteriochlorin has a 40–70% fluores-cence quantum yield depending on solvent, so it may be a goodfluorescent tag. The isobacteriochlorins also display enhancedtwo-photon absorption, thereby allowing the use of 860 nmlight to excite the compound. While the two-photon cross sec-tion of 25 GM units is not large, excitation of low chromophoreconcentrations can induce apoptosis. The glycosylated com-pounds accumulate in cancer cells and a head and neck squa-mous carcinoma xenograft tumor model in mice. Thesecompounds are robust to photobleaching.

INTRODUCTIONPorphyrinoids have been studied extensively for a variety ofapplications such as dyes for solar energy harvesting, catalystsand sensors for biological processes such as glucose transporta-tion (1). These macrocycles are also extensively studied as pho-todynamic therapeutic (PDT) agents for the treatment of a

variety of diseases such as cancer and infections (2–5). There issignificant research aimed to improve both the photophysical andbio-targeting properties of PDT agents (6,7). For both diagnosticand PDT applications, the use of the lower energy light is animportant photophysical property for next generation chromo-phores because red to near-infrared (IR) light penetrates deeperinto tissues (7–9). Reduced porphyrins have stronger and redderlowest energy absorption bands (10–12). Glycosylation targetsmany cancer types because of the significantly increased numberof glucose transporters and lectin-type receptors in the membrane(13–15). Carbohydrate–protein interactions are essential to alarge number of biological processes such as metastasis (16),receptor-mediated endocytosis, inflammation and infections bypathogens (13). As noted by Warburg, cancer cells consume glu-cose and produce lactic acid under aerobic conditions (17,18).There are many recent reports of glyco-photosensitizers (19).Nonhydrolyzable sugars appended to PDT agents such as por-phyrinoids minimizes cleavage of the targeting motifs byenzymes and low pH around cancer tissues, in lysosomes, and inendosomes (20–24).

Chlorins are porphyrinoids with one pyrrole double bondmissing, while isobacteriochlorins, and bacteriochlorins have twodouble bonds missing on the adjacent and opposite pyrroles,respectively. Oxidative and reductive transformations are used toform the natural and synthetic chromophores (10,25,26). Porphy-rins, chlorins, isobacteriochlorins and bacteriochlorins each haveunique photophysical properties that are exploited by nature (27)and can be used for diverse applications (1). The Goutermanfour-orbital model generally explains the consequences of reduc-ing the double bonds on the frontier molecular orbitals (28–31).For example, the intensity of the lowest energy UV–visibleabsorption band (Qy) in the red region progressively increasesand redshifts as the number of missing double bonds increases:porphyrins to chlorins to isobacteriochlorins to bacteriochlorins.The typical fluorescence quantum yield (Φf) for porphyrins, chlo-rins and bacteriochlorins is 0.10, 0.25 and 0.15 respectively (32).Metalation of the macrocycle further modulates the photophysi-cal properties, e.g. the enhanced spin-orbit coupling of Pd(II)

*Corresponding author email: cdrain@hunter.cuny.edu (Charles Michael Drain)†This paper is part of the Special Issue honoring the memory of Nicholas J. Turro.#A.A and S.T. contributed equally to this work.© 2013 The American Society of Photobiology

419

and Pt(II) complexes nearly quantitatively shunts the excitedstate into the triplet manifold (33–35) whereas Ni(II) complexesform nonluminescent metal centered d,d states (36,37). Thus,chlorins are widely studied as second-generation PDT agents(38,39), and recently bacteriochlorins are reported for this appli-cation (23,40–42). Conjugated exocyclic chalcones are anothermeans to extend the low-energy absorption into the red (43).

To understand the potential therapeutic or diagnostic value ofchromophores, the photophysical properties such as the fluores-cence quantum yield, singlet state lifetime, triplet quantum yieldand the quantum yield of singlet oxygen formation are evaluated(44). For therapies, red light absorption, efficient intersystemcrossing to the triplet manifold and suitable triplet lifetimes areneeded to maximize the photosensitized formation of singlet oxy-gen. Singlet oxygen is the initiation point for the formation ofreactive oxygen species that elicit cell stress and/or death fromoxidative damage at multiple sites, including mitochondria, endo-plasmic reticulum, and cell membranes (7). For luminescentdiagnostics and trackers for biochemical applications, the fluores-cence quantum yield should be optimized. A chromophore witha balance between the singlet and triplet quantum yields canpotentially be used for both detection and treatment (theragnos-tic), but must have efficient and robust targeting motifs.

Two-photon absorption (2PA) is the simultaneous absorptionof two low-energy photons to produce the same excited state ofthe dye as when excited with a single photon of twice the energy(45). 2PA fluorescence microscopy technologies are being devel-oped for biochemical studies, pathology and imaging applications(46,47). Chromophores with significant 2PA cross sections areanother avenue toward using red light to photosensitize the for-mation of singlet oxygen (48). Because red light penetrates dee-per into tissues, endogenous biomolecules have a small 2PAcross section and the high light flux needed to simultaneouslyabsorb two photons, the phototoxicity is confined to the narrowarea where the light flux is greatest (~0.1 mm2) (49,50).

Most porphyrin derivatives have small 2PA cross sections(45,51). Peripherally appended 2PA dyes can transfer energy toa porphyrin core (52–54). Ethyne or butadiyne-bridged porphyrindimers and oligomers (55–57), self-assembled ethyne or but-adiyne-bridged arrays (58,59) and aggregates (60,61) are reportedto have large 2PA cross sections. Some water soluble and gly-cosylated yne-linked 2PA systems are also reported (48,62–64).Some of these systems may also have significant single-photonabsorptions in the same near-IR region (65). The large molecularsize of these systems may be a limiting factor in their practicalapplications as PDT agents.

We reported that 5,10,15, 20-tetrakis(2,3,4,5,6-pentafluorophe-nyl)porphyrin (TPPF20) can serve as a core platform to make non-hydrolyzable glycosylated porphyrins (20–22) and solution phasecombinatorial libraries appended with a variety of bio-targetingmotifs (66,67). The nonhydrolyzable tetraglucose derivative(PGlc4) is a quite selective and effective PDT agent in vitro studiesusing several cancer cell lines, such as MDA-MB-231 humanbreast cancer cells (21,22). Low light irradiation and <1 μM por-phyrin concentrations induce apoptosis predominantly by damageto the endoplasmic reticulum (68). TPPF20 is readily modified toform the corresponding chlorin (ChlF20), isobacteriochlorin(IbacF20) and bacteriochlorin (BacF20) (69). We recently reportedthat these can be thioglycosylated in high yield to form CGlc4,IGlc4 and BGlc4 (Scheme 1) using the same click-type chemistryand that these are also taken up by cancer cells (23,24).

Herein, we describe the photophysical properties of this seriesof porphyrinoids. The overall goal is to assess their potential inapplications such as biomarkers and therapeutics under single-photon and two-photon excitation conditions. The resultspresented here suggest that a single porphyrin dye molecule, asopposed to a multichromophoric system, can possess a sufficienttwo-photon cross section to be a good two-photon imagingagent. Specifically, IbacF20 and IGlc4 have a sufficient 2PA andemission to obtain high-quality two-photon microscopy images.These results also suggest that IGlc4 is a good candidate forsimultaneous imaging and PDT using near-IR light. Because ofsignificantly stronger Q bands at 730 nm and high triplet quan-tum yield, BGlc4 has good potential as a PDT agent under sin-gle-photon conditions. The CGlc4 has intermediate photophysicalproperties that may be used for both detection and ablation ofcancer.

MATERIALS AND METHODSTPPF20 was purchased from Huhu Technology or Frontier Scientific andused as received; solvents from Fisher Scientific were purified by stan-dard methods and reagents from Sigma-Aldrich.

UV–visible spectroscopy. The UV–visible spectra were recorded on aVarian Bio3 spectrophotometer using 1 cm cuvettes. The UV–visible datafor the glycosylated conjugates were reported earlier (23) and are in theSupporting Information for clarity. The concentrations of each solutionwere ca. 1 μM.

Fluorescence spectra and quantum yield. Fluorescence spectra andtime-correlated single-photon counting (TCSPC) were recorded with aFluorolog s3, Jobin-SPEX Instrument S.A., Inc. (23) on ~1 μM solutionsin ethanol and ethylacetate. Samples were excited at the same band(~509 nm) where absorbencies ≤0.1 and were within 10% of each other.For emission spectra, both the excitation and detection monochromatorshad a band pass of 2 nm. The corrected emission (for instrumentresponse) and absorption spectra were used to calculate the quantumyield. Fluorescence quantum yields were determined for these nongly-cosylated F20 analogues in solution relative to meso-tetraphenylporphyrin(TPP) in toluene, which has a fluorescence quantum yield of 0.11(70,71).

Fluorescence lifetime. TCSPC fluorescence lifetime of nonglycosylat-ed F20 and glycosylated conjugates was determined in three different sol-vents: ethanol, ethylacetate and phosphate-buffered saline (PBS). A405 nm NanoLED laser, average power 13.6 pJ per pulse with a sourcepulse width <200 ps was used to excite the molecules. The instrumenthas 200 ps resolution. The data were fit using the instrument software.

Photobleaching. Photostability was assayed by exposing 1 μM

solutions of these conjugates in ethanol to sun light with a power of 40–80 W m�2 for about 2 h and taking the UV–visible spectra (see SupportingInformation).

Octanol/water partition coefficient. The octanol/water partition coeffi-cients were determined by saturating 1:1 (v/v) mixtures of the solventswith the thioglycosylated porphyrinoids. We shook the mixture well andwaited for 8–10 h to assure the two layers were separated completely.UV–visible spectra of the two layers were then taken to measure theSoret (B) and first Q-band intensities of the thioglycosylated compounds.These data were confirmed by saturating each solvent separately beforemixing equal volumes (23).

Confocal microscopy. To check the photostability of CGlc4 in cells,confocal microscopic measurements were done. K:Molv NIH 3T3 cellswere plated onto coverslips in cell culture dishes. CGlc4 dissolved inmethanol was added to the cultures to a final concentration of 0.1 lM.After incubation for 24 h, the cells were rinsed three times with PBS andincubated with a 4% paraformaldehyde solution for 15 min at 37°Cunder cell growth conditions. Cells were then washed three times withPBS, mounted in Dako fluorescence mounting medium and visualizedusing a Zeiss LSM510 laser scanning confocal microscope. The cellswere exposed to the laser light and images were captured. An oil immer-sion objective lens (Leica, Fluor-40X, NA 1.25) was used for cell imag-ing. Images were captured with excitation at 552 nm and the emissionband pass filter was 578–700 nm. The two-photon microscope was used

420 Amit Aggarwal et al.

to investigate the photostability of IbacF20 on the same cell line underthe identical cell culture conditions. The compound was excited at860 nm and emission was recorded between 500 and 670 nm.

Two-photon imaging. Two-photon imaging used a custom-built mul-tiphoton imaging system at Weill Cornell Medical College. The systemconsists of an Olympus BX61 upright microscope and a BioRad 1024scan head. The specimens were excited using a tunable Ti-Sapphire laser(Mai Tai, Spectra-Physics) tuned to 860 nm. The laser power under theobjective is controlled through a Pockel Cell (Conoptics). To avoid one-photon process, 860 nm excitation light was used and the emitted lightcollected was between 500 and 670 nm. Note that the lowest energy opti-cal absorption bands for the compounds in aqueous media are as follows:PGlc4 645 nm, CGlc4 649 nm, IGlc4 645 nm and BGlc4 730 nm. Thus,under these conditions, there is no single-photon excitation of the com-pounds.

Two-photon cross section. 2PA measurements were done as previ-ously described (72). Briefly, a Ti–sapphire laser (Spectra-Physics Tsu-nami) pumped by a frequency-doubled diode-pumped solid-state laser

(Spectra-Physics Millennia) provided 90 fs pulses operating at 82 MHzwas used to probe the 2PA properties of the dyes, and a continuous wave(CW) laser was used for the single-photon studies. Both lasers were oper-ated at the same power (6 mW). Experiments concerning quadraticdependence with the laser power were obtained using the femtosecond(fs) laser. Experiments were performed under the same conditions withrhodamine 6G as a standard and calculations used an existing protocol(73,74).

RESULTS AND DISCUSSIONThe synthesis of ChlF20, IbacF20 and BacF20 starts from thecommercially available TPPF20 and cycloaddition reactionsacross pyrrole double bonds, as reported by Cavaleiro et al. (69).The corresponding thioglycosylated conjugates, PGlc4, CGlc4,IGlc4 and BGlc4, are made by efficient nucleophilic substitution

N

NH

N

HN

F

F

F

FF

FF F

F

F

F

FF

F

F

FFF

F

F

N

CH3

N

CH3

N

NH

N

HN

F

FF

F

F

F F

F

F

F

FF

F

F

F

FF

F

F

F N

CH3

HN

N

NH

N

F

F

F

F F

F

FF

F

F

F

F

F F

F

F

FF

F

FTPPF20, (1)

ChlF20, (2)

IbacF20, (3)

BacF20, (4)

N

NH

N

HN

F

F

F

F

F

F

F

F F

F

F

F

F

F

F

SRF

N

CH3

SRRS

RS

NH

N

NHN

F

F

FFF

F

FF

F

F

F

F

FRS

F

N CH3

SR

SRRS N

CH3

Glc, R = OHOHO OH

OH

N

NH

N

HN

F

F

FF

F

F

FF F

F

F

F

F

F

F

SRF

N

CH3SRRS

RS N

CH3

CGlc4, (6)

IGlc4, (7)

BGlc4, (8)

N

NH

NHN

F

F

F

FF

F

F

F

FF

F

F

F

F

FRS

F

SR

SRRS

PGlc4, (5)

F

FNH

N

NHN

F

F

FFF

F

FF

F

F

F

F

FF

F

N CH3

F

FF N

CH3

F

F

Scheme 1. Structure of nonglycosylated (1–4) and glycosylated (5–8) conjugates of porphyrin. The syn vs anti compounds for the bacteriochlorins andisobacteriochlorins are isolated, but the diastereomers for CGlc4 and two sets of diastereomers for IGlc4 and BGlc4 (see Ref. (23)) are not separated forthese studies.

Photochemistry and Photobiology, 2014, 90 421

of the F group at the para position of the TPPF20 (20,21,23,67).Other click-type reactions can yield glycosylated porphyrins(75). The reduction of pyrrolic double bonds and the methylpyrr-olidine moiety used to stabilize the chlorin, isobacteriochlorinand bacteriochlorin may cause a deviation from the planaritycompared to the TPPF20 molecule, which may further change inthe energy gap between highest occupied molecular orbital(HOMO) and lowest unoccupied molecular orbital (LUMO) ofthe porphyrinoids. The classical four-orbital model developed byGouterman account for the major optical features of the free baseand metalloporphyrinoids in terms of transition between HOMO-LUMO (76). In addition to the rearrangement of the molecularorbitals due to the reduced double bonds, the redshift of the opti-cal bands can also arise from the distortions in the macrocycle(36,37).

UV–visible

The electronic spectra for ChlF20, IbacF20, BacF20, PGlc4, CGlc4,IGlc4 and BGlc4 were recorded in three different solvents: ethyl-acetate, ethanol and phosphate-buffered saline (PBS, pH = 7.4)over the wavelength range 300–900 nm (the upper limit of theinstrument). UV–visible absorption spectra of compoundsChlF20, IbacF20 and BacF20 in ethylacetate are shown in Fig. 1.The UV–visible absorption spectra of ChlF20, IbacF20 andBacF20 are slightly blueshifted from the corresponding thiogly-cosylated conjugates, CGlc4, IGlc4 and BGlc4. TPPF20 is some-what different from PGlc4. The shift in the peaks may arisebecause of the change in the electronic environment by substitu-tion of the electron withdrawing para F-atom on the meso-substi-tuted perfluorophenyl group by the electron donating S-atom ofthe thioglucose. The Soret (B) bands for compounds ChlF20,IbacF20, and BacF20, and for conjugates CGlc4, IGlc4 and BGlc4broaden and are blueshifted compare to the parent TPPF20 andPGlc4. For IbacF20 and IGlc4, the Soret band has two shoulderson the each side of the kmax, whereas for compounds BacF20 andBGlc4, the Soret band splits into two peaks. A significantincrease in the absorption intensity of the lowest energy Q-bandfor ChlF20, IbacF20 and BacF20 and for conjugates CGlc4, IGlc4and BGlc4 were observed compared to the parent porphyrinsTPPF20 and PGlc4. The lower energy Q-band for ChlF20 and

CGlc4 at 649 nm in ethylacetate has about 25-fold greater inten-sity than the corresponding Q-band of TPPF20 at 632 nm. ForIbacF20 and IGlc4, a 5-fold increase in the absorption intensitywas observed at 647 nm compared to TPPF20 at 632 nm,whereas for BacF20 and BGlc4 the lowest energy Q-band is red-shifted and appears at 730 nm with ca. 70-fold increased inten-sity compared to the absorption of TPPF20 at 632 nm and PGlc4at 649 nm. For all of these compounds, decomposition productssuch as dipyrrole methane or billirubin were not observed evenupon exposure to ambient light. Also, there is no absorptionpeaks observed between 750 and 900 nm, in the area of two-photon activity for the nonglycosylated IbacF20 compound. UV–visible spectral data of compounds ChlF20, IbacF20 and BacF20in different solvents are summarized in Table 1, whereas thespectra for compounds PGlc4, CGlc4, IGlc4 and BGlc4 werereported earlier by us (23) (see Supporting Information).

Fluorescence

The fluorescence emission spectra for ChlF20, IbacF20, BacF20,PGlc4, CGlc4, IGlc4 and BGlc4 were recorded in three differentsolvents: ethylacetate, ethanol and PBS and are found to have astrong solvent dependence. The emission peaks for ChlF20,IbacF20 and BacF20 are given in Table 1, whereas we earlierreported the spectra for compounds PGlc4, CGlc4, IGlc4 andBGlc4 (23) (see Supporting Information). Emission spectra ofcompounds ChlF20, IbacF20 and BacF20 in ethylacetate areshown in Fig. 2. There are negligible Stokes shifts observed forthese conjugates in different solvents. The small Stokes shift for

Table 1. Absorption and emission spectral data of non-glycosylated F20porphyrinoids conjugates.

Comp. Solvent UV–visible (nm) Emission (nm)

ChlF20 Ethanol 399, 502, 528, 597, 651 654, 708Ethylacetate 398, 501, 527, 597, 649 655, 709

IbacF20 Ethanol 381, 504, 541, 583, 635 592, 645, 707Ethylacetate 380, 503, 541, 581, 647 594, 645, 711

BacF20 Ethanol 348, 374, 503, 729 647, 723Ethylacetate 347, 373, 502, 729 651, 735

Figure 2. Emission spectra of compounds 1–4 in ethylacetate; excitationat 509 nm where the O.D was 0.014 for each compound.

Figure 1. UV–visible absorption spectra of compounds 1–4 in ethylace-tate in 1 cm cuvettes and the concentration of each solution was 1 μM.

422 Amit Aggarwal et al.

PGlc4 and CGlc4 in the three solvents likely indicates that mostof the specific solvent–solute interactions are related to the sugarmoieties. Notably, for the IGlc4 in PBS, the strongest fluores-cence band maxima are at 606 nm with a much weaker emissioncentered at 650 nm (see Supporting Information). Excitationspectra indicate the presence of only the given compounds. Thefluorescence quantum yield for ChlF20, IbacF20, BacF20, PGlc4,CGlc4, IGlc4 and BGlc4 and in the above-mentioned solvents areshown in Table 2. The 0.17 and 0.36 fluorescence quantumyields for CGlc4 and for IGlc4 in PBS are 6-fold and 12-foldgreater than the porphyrin analogue PGlc4. There is a smalldecrease in fluorescence quantum yield upon replacement of thepara F-atom by the thioglucose due to both electronic and heavyatom effects. The greater fluorescence quantum yield of CGlc4compared to m-THPC (Φ = 0.09 in methanol) (38), likely arisesfrom the presence of the 16 F groups. Interestingly, the BGlc4system with the 730 nm absorption band has a fluorescencequantum yield of about 0.05 and thus for the present we assumemost of the excited state intersystem crosses to the triplet mani-fold. Nonetheless, the fluorescence is sufficient to observe theuptake into cancer cell lines. Because BGlc4 is a minor product,the smaller anti/syn ratio, and the set of diastereomers, this com-pound was not extensively evaluated. Our initial hypothesis wasthat the N-methylpyrrolidine moieties used to make and stabilizethe chlorins, bacteriochlorins and isobacteriochlorins would haveminimal effect on cell uptake because these are sandwichedbetween the phenyl sugars; however, the octanol/water partitioncoefficients range from ca. 10 for IGlc4 and BGlc4 to 30 forCGlc4 and 40 for PGlc4 (23).

Excited state decay

The decay of the electron from the excited singlet state, S1 to theground singlet state, S0 follows a series of radiative and/or nonra-diative processes. The rate constant for these decay processes

relates to the fluorescence quantum yield (Uf) and the fluores-cence lifetime (sf) can be calculated using following equations:

sf ¼ 1=ðkf þ kic þ kiscÞ ð1Þ

Uf ¼ kf=ðkf þ kic þ kiscÞ ð2Þ

kf, kic and kisc are the rate constants for the radiative spontaneousfluorescence, internal conversion and intersystem crossing to thetriplet excited state, respectively. Using the above equations, therate constant for nonradiative decay, knr is calculated (see Sup-porting Information). These compounds exhibit different degreesof excited state decay via radiative, nonradiative internal conver-sion, and S1 ? T1 intersystem crossing pathways that are alsosolvent dependent. A high fluorescence quantum yield indicatesradiative S1 ? S0 decay dominates, whereas a low fluorescencequantum yield suggests decay is predominantly nonradiative viainternal conversion and S1 ? T1 (33,41,77). The greater Uf andradiative decay time constants and low time constant for nonradi-ative decay for CGlc4 and IGlc4 indicate that these conjugatesmay serve as fluorescent tags. The low Uf, the 10- to 20-foldsmaller radiative decay time constants, and the two-fold greaternonradiative decay time constant for PGlc4 and BGlc4 indicatethat these compounds enter the triplet manifold more efficiently,thus can serve as PDT agents.

The macrocycle distortion from the planarity and increasedconformational dynamics causes the destabilization of the p-sys-tem and contributes to the significantly shorter lifetimes (78).These effects contribute to the ca. two- to four-fold shorter life-times for CGlc4, IGlc4 and BGlc4 conjugates compared to theplanar TPPF20 compound.

The glycosylated derivatives are used to evaluate uptake byK:Molv NIH 3T3 mouse fibroblasts cells, vide infra. Ethylace-tate, PBS, methanol or ethanol solvents were used to probe thephotophysical properties as the amphipathic glycoporphyrinoids

Table 2. Photophysical data for compounds 1–8.

Compound Solvent Φf (±SD) sf ns (v2) kf (ns)

�1 (kf)�1 ns knr (ns)

�1 (knr)�1 ns

TPPF20 (1) EA 10.08 (1.21)EtOH 11.14 (1.04)

ChlF20 (2) EA 0.34 (0.05) 7.06 (1.21) 0.048 20.8 0.094 10.6EtOH 0.48 (0.06) 7.48 (1.37) 0.064 15.6 0.070 14.3

IbacF20 (3) EA 0.60 (0.03) 5.38 (1.25) 0.112 8.9 0.074 13.5EtOH 0.72 (0.01) 3.52 (1.19) 0.205 4.9 0.079 12.7

BacF20 (4) EA 0.062 (0.005) 6.80 (1.41) 0.009 111.1 0.138 7.3EtOH 0.07 (0.003) 6.88 (1.30) 0.010 100.0 0.135 7.4

PGlc4 (5) EA 0.052 (0.004) 9.88 (1.05) 0.0053 188.7 0.096 10.4EtOH 0.05 (0.002) 10.84 (1.24) 0.0046 217.4 0.088 11.4PBS 0.03 (0.002) 10.66 (1.28) 0.0028 357.1 0.091 11.0

CGlc4 (6) EA 0.39 (0.04) 6.91 (1.42) 0.056 17.9 0.089 11.2EtOH 0.43 (0.05) 7.21 (1.34) 0.060 16.7 0.079 12.7PBS 0.17 (0.05) 6.84 (1.24) 0.025 40.0 0.121 8.3

IGlc4 (7) EA 0.60 (0.04) 5.93 (1.27) 0.101 9.9 0.068 14.7EtOH 0.70 (0.15) 4.29 (1.46) 0.163 6.1 0.070 14.3PBS 0.36 (0.07) 2.98 (1.47) 0.121 8.3 0.215 4.7

BGlc4 (8) EA 0.055 (0.003) 7.01 (1.32) 0.0078 128.2 0.135 7.4EtOH 0.047 (0.005) 6.96 (1.39) 0.0067 149.3 0.137 7.3PBS 0.03 (0.007) 5.44 (1.33) 0.0055 181.8 0.178 5.6

The quantum yield of conjugates 2–4 were calculated in two different solvents, ethanol (EtOH) and ethylacetate (EA), while phosphate buffered saline(PBS) was also used for 6–8. The molecules were excited at 509 nm in each case where the O.D. = 0.02. TPP was used as reference, Φf for TPP in tol-uene = 0.11. The lifetime of these compounds were measured under argon, by purging argon gas through the cuvette for ca. 10 min. The errors in thesinglet state lifetime were �200 ps, which is the lower limit of time measurement of our instrument.

Photochemistry and Photobiology, 2014, 90 423

may localize in aqueous, highly polar, or hydrophobic environ-ments within cells. The electronic spectra, fluorescence and fluo-rescence microscopy studies all show that these compounds arerobust to photobleaching (Fig. 3). The remaining 16 F groupsimpart stability toward oxidative damage to the macrocycle andfurther enhance the photonic properties. The significantly greaterfluorescence quantum yield of the IGlc4 system indicates that itcan be used at <25 nM concentrations in fluorescence micros-copy. For PDT applications, sensitizers with strong red absorp-tions are generally considered better because these wavelengthspenetrate deeper into tissue. The optical cross section of CGlc4in the red region is significantly larger than the porphyrin or iso-bacteriochlorin analogues. Thus, if red light is used to activatethe CGlc4 dye, the increased light absorptivity more than com-pensates the reduced triplet quantum yield. The enhanced fluores-cence quantum yield of IGlc4 makes it better suited for taggingand sensor applications, whereas the very strong 730 nm absorp-tion and the low fluorescence quantum yield of BGlc4 indicatethat this may be the best photosensitizer for PDT of the com-pounds described herein. The singlet oxygen quantum yields arereported in Table 3.

Photobleaching of CGlc4

K:Molv 3T3 NIH cells were incubated with CGlc4 for 24 h to afinal concentration of 0.1 μM and were prepared for confocalmicroscopy as described in the experimental section. The excita-tion wavelength was selected 552 nm and the emission band fil-ter was 578–700 nm. The images show almost nophotobleaching for this compound (Fig. 3), thus indicating that

this chromophore can also be a good imaging agent, whereas thecommercially available tracker dyes used for imaging purposesphotobleach quickly under the same conditions (79).

Two-Photon Microscopy

As the emission of IGlc4 and BGlc4 taken up by cancer cellswas observable by two-photon microscopy, we investigated thesederivatives to quantify the two-photon absorption properties.Similar to our previous studies on the PGlc4 with MDA-MB-231, Chinese hamster ovary cells were incubated overnight withPGlc4, CGlc4, or a 5:1 mixture of IGlc4 and BGlc4 at 10 lM.Only the 5:1 mixture of IGlc4 and BGlc4 is observable with two-photon microscopy, or at least, some fluorescence light in therange of 500–670 nm is detected when the compounds areexcited with 860 nm light (Fig. 4). These results clearly showthe possibility that either IGlc4 or BGlc4 has an appreciable two-photon cross section. To delineate the contributions of each com-pound to the observed two-photon microscopy images, the cellswere incubated under the same conditions with IbacF20 andBacF20 separately. UV–visible spectra show that neither com-pound absorbs at the 860 nm excitation used in the two-photonmicroscopy (Fig. 1). As the fluorescence of BacF20 beginsaround 720 nm and the detector is set up from 500 nm until670 nm, significant fluorescence of this compound is notobserved. The IbacF20 is two-photon active (Fig. 5).

Two-photon excitation

Although the one-photon absorption spectra of IbacF20 andBacF20 show no absorption peaks around 860 nm (Fig. 1), bothcompounds were excited using a CW laser and a fs laser toinsure that the absorption process at this wavelength is due to atwo-photon process. The lower irradiance of the CW laser typi-cally results in one-photon excitation, whereas the fs laser canresult in two-photon excitation (72). The two-photon efficiencydepends on the instantaneous irradiance. The powers of bothmodes used here were the same.

Rhodamine 6G (R6G) was used as two-photon reference tomeasure the two-photon activity of IbacF20 and BacF20. The

Figure 3. K:Molv 3T3 NIH cells plated onto coverslips were incubated with CGlc4 for 24 h, washed three times with phosphate-buffered saline (PBS)buffer, fixed with 4% paraformaldehyde, again washed three times with PBS and mounted in Dako fluorescence mounting medium. Confocal imageswere taken using excitation wavelength 552 nm and emission filter was 578–700 nm. A: image after first scan and B: image of the same sample after25 scans. The images are as collected.

Table 3. Singlet oxygen quantum yield (1O2ΦD).

Compound (1O2ΦD)(�SD)

PGlc4 0.85 (0.04)CGlc4 0.32 (0.05)BGlc4 0.28IGlc4 0.59 (0.09)

All the compounds were excited at 532 nm in methanol-d1 solvent; datataken from ref. (23).

424 Amit Aggarwal et al.

two-photon reference, R6G, is not excited when the CW laser isused but is excited and fluorescence is measured when a fs laseris used. The fs pulse for IbacF20 indicates that IbacF20 is two-photon active, and one-photon absorption at 860 nm is notobserved. Mixed results were obtained when BacF20 is excitedusing a similar setup as one-photon absorption can be observedwhen 760, 780 or 800 nm wavelengths are used. Whereas atlower energy wavelengths (820, 840, 860 and 880 nm), bothtwo-photon and one-photon absorption were observed (Support-ing Information).

Two-photon absorbance processes imply that the fluorescenceemission increases quadratically with increasing laser power,rather than linearly for one-photon emission. This behavior arisesfrom the fact that two photons are absorbed in the same quantumevent. With the fs laser, BacF20, IbacF20 and R6G were excitedat 860 nm, and the fluorescence light measured as a function oflaser power (Fig. 6). The observed combination of both absorp-tion processes in the case of BacF20 when laser excitation at760–800 nm likely arises from the small but long tail of thelower energy absorption peak kmax 730 nm, or there is a verysmall band centered at ca. 830 nm. An anti-Stokes effect is alsopossible wherein the molecule gets vibrational and rotationalexcitation from the basal level from kt energy, thus allowingexcitation of the molecule with a lower energy than normallyexpected. Neither the small tail or the absorption band nor theanti-Stokes effect is observed with the IbacF20 compoundbecause its lowest energy absorption peak kmax at 600 nm is faraway from the 760 nm laser. Thus, IbacF20 can only have a 2PAat wavelengths greater than 760 nm (Table 4).

Emission from IbacF20 has a clear quadratic dependence onthe laser power that is similar to the R6G standard while BacF20shows a linear dependence on the laser power (Fig. 6). Theseresults confirm that IbacF20 is indeed two-photon active, and that

Figure 4. Chinese hamster ovary (CHO) cells were incubated overnight with (a) 10 lM PGlc4, (b) 10 lM CGlc4, (c) 10 lM of a mixture of IGlc4: BGlc4(5:1). Two-photon microscope excitation light was at 860 nm, and the detector was set for 500–670 nm. The image data were collected for 1 s. Theimages are as collected.

Figure 5. K:Molv NIH 3T3 cells were incubated with 10 lM of IbacF20overnight. Two-photon microscope excitation light was at 860 nm, andthe detector was set for 500–670 nm. The image data were collected for1 s. The image is as collected.

Figure 6. Dependence of light emitted vs laser power. Excitation:860 nm. Light collected: 500–800 nm. squares = IbacF20, diamonds =rhodamine 6G, triangles = BacF20.

Table 4. Two photon cross section values of IbacF20.

Wavelength (nm) Cross section (GM)

760 1.7780 7.5800 3.2820 3.1840 3.2860 24.5880 6.1

Photochemistry and Photobiology, 2014, 90 425

BacF20 either has little or no two-photon absorption (2PA) or thesingle-photon processes dominate the observed fluorescence.

Two-photon cross sections are difficult to measure consis-tently and accurately because of the large flux of photons neededfrom the fs laser can vary significantly. For this reason, all the2PA calculations are based on the fluorescence light emittedwhen the sample is excited and by assuming this is directly pro-portional to the photons absorbed. This also assumes that thequantum yields of both reference and the sample are constant asa function of the wavelength. The reference for 2PA is R6G inmethanol at 110 lM (quantum yield = 0.93). IbacF20 was dis-solved in dimethylsulfoxide at the same concentration (quantumyield = 0.15). The cross section (r) of IbacF20 was calculatedfor each wavelength using the following equation:

rsðkÞ ¼ FðkÞ � QYr � nrFrðkÞ � QY � n

rrðkÞ

where F(k), Fr(k), QY, QYr, rs(k) and rr(k) are the two-photonfluorescence of the sample, two-photon fluorescence of the refer-ence, quantum yield of sample, quantum yield of the reference,sample cross section and the reference cross section respectively,nr and n are the refractive index of the solvents. The cross sec-tions obtained are expressed in Table 4. Two-photon cross sec-tions for molecular systems are expressed in Goeppert–Mayer(GM) units, where 1 GM is 10�50 cm4 s per photon.

Two-photon photobleaching of IbacF20 in cells

The photostability of the fluorescent imaging agent and/or PDTphotosensitizer is very important, since longer photostabilitywould allow imaging over a greater amount of time or multipleimages to be taken, and from a therapeutic perspective, the stabilityof the sensitizer to photobleaching increases the efficacy of the dye.

To investigate the photostability of IbacF20, the compoundwas incubated into K:Molv NIH 3T3 cells. After 24 h the cellswere washed three times with PBS buffer. A total of 25 scanswere performed using the two-photon microscope and the firstimage from the first scan was compared to the last image fromthe last scan. As shown in Fig. 7, IbacF20 does not significantlyphotobleach. Thus, this core platform may be an excellent choicefor both therapeutic and imaging applications where one needs

to follow cells or biochemical processes over time. The photosta-bility of IGlc4 and CGlc4 was also measured in sunlight onsunny days wherein 1 lM solution of the conjugates in ethanolwas exposed to sunlight and UV–visible spectra were recordedvs time for 3–4 h (see Supporting Information). Photobleachingwas observed for CGlc4 ca. 5%, for IGlc4 ca. 10%, and wereported previously ca. 10% for IGlc4 (23).

Bioimaging

Head and neck squamous carcinoma (HNSCC) tumors indicate apoor long-term prognosis. Single-photon fluorescence imaginginvestigations were made to determine the uptake of the glycosy-lated compounds into HNSCC in mouse models. Subcutaneousinjection of athymic mice with a HNSCC cell line expressinggreen fluorescent protein (GFP) results in tumors that can betracked by fluorescence imaging. When tumors reached adequatesize 100 lL of 5 mg mL�1 solution of PGlc4 or IGlc4 or CGlc4in PBS was injected in tail vein of the mouse (50). Fluorescenceimages were collected periodically using different band pass fil-ters to distinguish GFP vs dye emission (Fig. 8). This series ofwhole mouse images indicates some accumulation of the IGlc4and CGlc4 in the xenograft tumor. IGlc4 fluorescence was ini-tially observed in the mouse brain, spinal column and liver5 min after the tail vein injection, and a maximum IGlc4 fluores-cence was detected in the xenograft tumors after 30 min injec-tion. The IGlc4 fluorescence indicates that the dye was cleared inca. 21 h after injection. CGlc4 distribution was significantlydelayed compared to IGlc4 in that the fluorescence was initiallydetected in xenograft tumors, brain and liver 21 h after injection.Interestingly, a large amount of CGlc4 fluorescence was accumu-lated in the xenograft tumors 24 h after injection. PGlc4 does notyield consistent results under these conditions, which is likelydue to the low fluorescence quantum yield and perhaps becausethe dyes remain aggregated for a longer time in the mice. Micewere monitored an additional 21 days to assess the effects ofIGlc4, CGlc4 and PGlc4 on the xenograft tumor growth. BothCGlc4 and PGlc4 showed a notable capacity to inhibit xenografttumor growth under the standard conditions for keeping mice,i.e. no specific light irradiation of the tumors. The tumor weightin CGlc4- or PGlc4-treated groups was approximately 24–34%lower than untreated control groups (Fig. 9). This may indicate

Figure 7. K:Molv 3T3 NIH cells were incubated with IbacF20 for 24 h and rinsed three times with phosphate-buffered saline buffer, the two-photonmicroscopy was done repeatedly to examine the photostability of the compounds under these conditions. Excitation was at 860 nm, and detectionbetween 500 and 670 nm. The two-photon microscopic image after one scan (A), and the same sample after 25 scans (B). The images are as collected.

426 Amit Aggarwal et al.

some effect of the compound under the ambient light used tokeep the mice. Although the 1O2ΦD of 0.59 is greater than forCGlc4, there was no statistically significant difference of tumorweight between IGlc4-treated and untreated groups, which mayresult from the faster pharmacokinetics.

CONCLUSIONAs nucleophilic aromatic substitution of the para fluoro group israpid and efficient, these studies provide the methods to synthe-size and evaluate a wide range of exocyclic motifs for applica-tions ranging from photonic materials to therapeutics (67).Amphipathic compounds such as these glycosylated dyes are

often found to be more effective PDT agents (80,81). BothCGlc4 and IGlc4 have significantly enhanced fluorescence quan-tum yields compared to the PGlc4, whereas the BGlc4 derivativehas a fluorescence quantum yield that is similar to the parentporphyrin. Thus, IGlc4 has greatest potential use as fluorescenttags, diagnostics, or imaging agents. The intermediate fluores-cence of the CGlc4 system may well serve as a theragnosticagent for targeting, detecting and treating diseased tissues. TheBGlc4 system has near optimal properties as the PDT agent, butnew synthetic strategies are needed to make these compounds.The much lower concentrations of CGlc4 and IGlc4 needed forfluorescence microscopy indicate that these compounds can beused as bioimaging and diagnostic tools. IbacF20 has 2PAbetween 760 and 880 nm, while BacF20 is largely a one-photondye that may have small contributions from two-photon pro-cesses. Both the compounds have advantages compared to cur-rently used photosensitizers for PDT: (1) strong red lightabsorbance, (2) the exocyclic glucose moieties target cancer cellsover normal cells. The 2PA properties of IbacF20, while not largecompared to the multichromophore systems reported, are suffi-cient for imaging applications and perhaps for therapeutic appli-cations. The role of aggregation and possible differences inmetabolism in vivo is under investigation.

Acknowledgements—We thank Dr. Tymish Y. Ohulchanskyy of theInstitute of Photomedicine, State University of New York at Buffalo forhelp in determining the two-photon cross section, supported by the U.S.National Science Foundation (NSF CHE-0847997 and 1213962 to CMD,and Hunter College science infrastructure is supported by the NationalInstitutes of Health, including the National Institute on Minority Healthand Health Disparities (8G12 MD007599) and the City University ofNew York. The RCMI/NIMHD program (8G12 MD007597) at HowardUniversity and The RTRN/NIMHD program (U54MD008149).

Figure 8. IGlc4 and CGlc4 uptake, distribution, clearance. Four-week-old, male Balb/c athymic nude mice (Nu/Nu) were obtained from Harlan SpragueDawley, Inc. (Indianapolis, IN). Mice were housed in temperature-controlled rooms (74 � 2°F) with a 12-h alternating light-dark cycle. Approximately3 9 106 GFP-JHU-22 cells in log phase were injected subcutaneously into the mice. The uptake experiments were done after 7 days after GFP-JHU-22cell inoculation. The mice received tail vein injections with a total volume of 100 lL of 5 mg mL�1 IGlc4 or CGlc4. The fluorescence images weretaken at the times indicated after injection. Each individual group has two images where the emission due to GFP is on the left of each time interval,and monitored using a 465–520 band pass filter. A 605–660 nm band pass filter was used to image the tumors with CGlc4 and IGlc4. The arrows indi-cated the location of tumor xenografts. A Xenogren IVIS instrument (Caliper Life Sciences, Hopkinton, MA) was used to collect these images.

Figure 9. Decrease in tumor weight after 21 days.

Photochemistry and Photobiology, 2014, 90 427

SUPPORTING INFORMATIONAdditional Supporting Information may be found in the onlineversion of this article:

Figure S1. UV–visible spectra of the compounds, 2 lM inethanol.

Figure S2. UV–visible spectra of the compounds, 2 lM inethylacetate.

Figure S3. UV–visible spectra of the compounds, 2 lM inPBS.

Figure S4. Emission spectra of the compounds in ethanol;excitation at 512 nm where the O.D. is ca. 0.05 for each com-pound.

Figure S5. Emission spectra of the compounds in ethylace-tate; excitation at 509 nm where the O.D. was 0.098 for eachcompound.

Figure S6. Emission spectra of the compounds in PBS; exci-tation at 512 nm, where the O.D. was 0.018 for each compound.

Figure S7. UV–visible spectra of the compounds, 1 lM inethanol.

Figure S8. Emission spectra of the compounds in ethanol;excitation at 509 nm where the O.D was 0.02 for all the com-pounds.

Figure S9. Changes in 1O2 intensity as a function of OD at532 nm. The lower ΦD from CGlc4 may partially due to the inef-ficient light absorption at 532 nm compared to IGlc4.

Figure S10. Emission spectra of IGlc4 in ethanol in air in aclosed vial show only a small degree of photobleaching of IGlc4in sun light with time.

Figure S11. UV–visible spectra for photobleaching of CGlc4in sunlight. 1 lM solution in ethanol.

Figure S12. Emission spectra for photobleaching of CGlc4 insunlight. 1 lM solution in ethanol.

Figure S13. UV–visible spectra of the BacF20 compound,1 lM in ethanol.

Figure S14. UV–visible spectra of the IbacF20 compounds,1 lM in ethanol.

Figure S15. K:Molv NIH 3T3 cells were incubated with thethree compounds (a) PGlc4, (b) CGlc4 and (c) IGlc4 to a finalconcentration of 10 lM for ca. 20–24 h. The cells were thenwashed with PBS three times to remove the unbound dye fromthe cells. Fluorescence images of the cells were then taken underthe identical microscopic conditions and were not enhanced,exposure time 0.1 s and magnification 30X.

Figure S16. Quantification of the dyes take up by the K:MolvNIH 3T3 cell line. Note that the ca. eight-fold increased fluores-cence quantum yield for CGlc4 in ethanol accounts for some ofthe increased signal, but the increase is much less than eight-foldso the data indicate that CGlc4 is not taken up by the cells asmuch as PGlc4. Similarly, the ca. 13-fold increase in the fluores-cence quantum yield for IGlc4 means that this compound is nottaken up as well as the PGlc4 but about the same as CGlc4. Thedifferences in octanol/water partition coefficient may account forthese differences in uptake.

REFERENCES

1. Kadish, K., K. Smith and R. Guilard (2010) The Handbook of Por-phyrin Science With Applications to Chemistry, Physics, Materials

Science, Engineering, Biology and Medicine. World Scientific Pub-lishers, Singapore.

2. Bonnett, R. (1995) Photosensitizers of the porphyrin the phthalocya-nine series for phtodynamic therapy. Chem. Soc. Rev. 24, 19–33.

3. Pandey, R. K. (2000) Recent advances in photodynamic therapy. J.Porphyrins Phthalocyanines 4, 368–373.

4. Sternberg, E. D., D. Dolphin and C. Br€uckner (1998) Porphyrin-based photosensitizers for use in photodynamic therapy. Tetrahedron54, 4151–4202.

5. Lovell, J. F., T. W. B. Liu, J. Chen and G. Zheng (2010) Activatablephotosensitizers for imaging and therapy. Chem. Rev. 110, 2839–2857.

6. Allison, R. R., G. H. Downie, R. Cuenca, X.-H. Hu, C. J. H. Childsand C. H. Sibata (2004) Photosensitizers in clinical PDT. Photodiag.Photodyn. Ther. 1, 27–42.

7. Agostinis, P., K. Berg, K. A. Cengel, T. H. Foster, A. W. Girotti, S.O. Gollnick, S. M. Hahn, M. R. Hamblin, A. Juzeniene, D. Kessel,M. Korbelik, J. Moan, P. Mroz, D. Nowis, J. Piette, B. C. Wilsonand J. Golab (2011) Photodynamic therapy of cancer: An update. CA– Cancer J. Clin. 61, 250–281.

8. Pushpan, S. K., S. Venkatraman, V. G. Anand, J. Sankar, D. Par-meswaran, S. Ganesan and T. K. Chandrashekar (2002) Porphyrinsin photodynamic therapy – A search for ideal photosensitizers. Curr.Med. Chem. – Anti-Cancer Agents 2, 187–207.

9. Ethirajan, M., Y. Chen, P. Joshi and R. K. Pandey (2011) The roleof porphyrin chemistry in tumor imaging and photodynamic therapy.Chem. Soc. Rev. 40, 340–362.

10. Spikes, J. D. (1990) New trends in photobiology: Chlorins as photo-sensitizers in biology and medicine. J. Photochem. Photobiol. B:Biol. 6, 259–274.

11. Senge, M. O. and J. C. Brandt (2011) Temoporfin (Foscan�,5,10,15,20-tetra(m-hydroxyphenyl)chlorin)—A second-generationphotosensitizer. Photochem. Photobiol. 87, 1240–1296.

12. Arnaut, L. G. (2011) Design of porphyrin-based photosensitizers forphotodynamic therapy. In Adv. Inorg. Chem., Vol. 63 (Edited by E.Rudi van and S. Gra_zyna), pp. 187–233. Academic Press, Amster-dam.

13. Ambrosi, M., N. R. Cameron and B. G. Davis (2005) Lectins: toolsfor the molecular understanding of the glycocode. Org. Biomol.Chem. 3, 1593–1608.

14. Dwek, R. A. (1996) Glycobiology: Toward understanding the func-tion of sugars. Chem. Rev. 96, 683–720.

15. Monsigny, M., A.-C. Roche, C. Kieda, P. Midoux and A. Obrenovitch(1988) Characterization and biological implications of membrane lec-tins in tumor, lymphoid and myloid cells. Biochimie 70, 1633–1649.

16. Gorelik, E., U. Galili and A. Raz (2001) On the role of cell surfacecarbohydrates and their binding proteins (lectins) in tumor metasta-sis. Cancer Metastasis Rev. 20, 245–277.

17. Warburg, O. (1956) On the origin of cancer cells. Science 123, 309–314.

18. Kim, J.-W. and C. V. Dang (2006) Cancer’s molecular sweet toothand the Warburg effect. Cancer Res. 66, 8927–8930.

19. Ballut, S., D. Naud-Martin, B. Loock and P. Maillard (2011) A strat-egy for the targeting of photosensitizers. Synthesis, characterization,and photobiological property of porphyrins bearing glycodendrimericmoieties. J. Org. Chem. 76, 2010–2028.

20. Pasetto, P., X. Chen, C. M. Drain and R. W. Franck (2001) Synthe-sis of hydrolytically stable porphyrin – and -glycoconjugates in highyields. Chem. Commun. 81–82.

21. Chen, X., L. Hui, D. A. Foster and C. M. Drain (2004) Efficientsynthesis and photodynamic activity of porphyrin-saccharide conju-gates: Targeting and incapacitating cancer cells. Biochemistry 43,10918–10929.

22. Chen, X. and C. M. Drain (2004) Photodynamic therapy using car-bohydrate conjugated porphyrins. Drug Design Rev. – Online 1. doi:10.2174/1567269043390988.

23. Singh, S., A. Aggarwal, S. Thompson, J. P. C. Tom�e, X. Zhu, D.Samaroo, M. Vinodu, R. Gao and C. M. Drain (2010) Synthesis andphotophysical properties of thioglycosylated chlorins, isobacterio-chlorins, and bacteriochlorins for bioimaging and diagnostics. Bio-conjug. Chem. 21, 2136–2146.

24. Drain, C. M., S. Singh, D. Samaroo, S. Thompson, M. Vinodu andJ. P. C. Tome (2009) New porphyrin glyco-conjugates. Proc. Soc.Photo-Opt. Instr. Eng. SPIE 7380, 73902K-1–73902K-9.

428 Amit Aggarwal et al.

25. Kadish, K. M., K. M. Smith and R. Guilard (2000) The PorphyrinHandbook: Synthesis and Organic Chemistry, Vol. 1. AcademicPress, San Diego.

26. Lim, S. H., H. B. Lee and A. S. H. Ho (2011) A new naturallyderived photosensitizer and its phototoxicity on head and neck can-cer cells. Photochem. Photobiol. 87, 1152–1158.

27. Mauzerall, D. C. (1998) Evolution of porphyrins. Clin. Dermatol.16, 195–201.

28. Gouterman, M. (1961) Spectra of porphyrins. J. Mol. Spect. 6, 138–163.

29. Ghosh, A. (1998) First-principles quantum chemical studies of por-phyrins. Acc. Chem. Res. 31, 189–198.

30. Yang, E., C. Kirmaier, M. Krayer, M. Taniguchi, H.-J. Kim, J. R.Diers, D. F. Bocian, J. S. Lindsey and D. Holten (2011)Photophysical properties and electronic structure of stable,tunable synthetic bacteriochlorins: Extending the features ofnative photosynthetic pigments. J. Phys. Chem. B 115, 10801–10816.

31. Taniguchi, M., D. L. Cramer, A. D. Bhise, H. L. Kee, D. F. Bocian,D. Holten and J. S. Lindsey (2008) Accessing the near-infrared spec-tral region with stable, synthetic, wavelength-tunable bacteriochlo-rins. New J. Chem. 32, 947–958.

32. Drain, C. M. and S. Singh (2010) Combinatorial chemistry of por-phyrins. In The Handbook of Porphyrin Science with Applications toChemistry, Physics, Materials Science, Engineering, Biology andMedicine, Vol. 2 (Edited by K. Kadish, R. Guilard and K. Smith),pp. 485–540. World Scientific, Singapore.

33. Kee, H. L., J. Bhaumik, J. R. Diers, P. Mroz, M. R. Hamblin, D. F.Bocian, J. S. Lindsey and D. Holten (2008) Photophysical character-ization of imidazolium-substituted Pd(II), In(III), and Zn(II) porphy-rins as photosensitizers for photodynamic therapy. J. Photochem.Photobiol. A: Chem. 200, 346–355.

34. Hirohara, S., M. Obata, H. Alitomo, K. Sharyo, T. Ando, S. Yanoand M. Tanihara (2009) Synthesis and photocytotoxicity of S-glu-cosylated 5,10,15,20-Tetrakis(tetrafluorophenyl)porphyrin metal com-plexes as efficient 1O2-generating glycoconjugates. Bioconjug. Chem.20, 944–952.

35. Hirohara, S., M. Obata, H. Alitomo, K. Sharyo S.-I.Ogata, C. Ohtsu-ki, S. Yano, T. Ando and M. Tanihara (2008) Structure and photo-dynamic effect relationships of 24 glycoconjugated photosensitizersin HeLa cells. Biol. Pharm. Bull. 31, 2265–2272.

36. Drain, C. M., S. Gentemann, J. A. Roberts, N. Y. Nelson, C. J.Medforth, S. L. Jia, M. C. Simpson, K. M. Smith, J. Fajer, J. A.Shelnutt and D. Holten (1998) Picosecond to microsecond photody-namics of a nonplanar nickel porphyrin: Solvent dielectric and tem-perature effects. J. Am. Chem. Soc. 120, 3781–3791.

37. Drain, C. M., C. Kirmaier, C. J. Medforth, J. Nurco, K. M. Smithand D. Holten (1996) Dynamic photophysical properties of conform-ationally distorted nickel porphyrins.1. Nickel(II) dod-ecaphenylporphyrin. J. Phys. Chem. 100, 11984–11993.

38. Bonnett, R., P. Charlesworth, B. D. Djelal, S. Foley, D. J. McGarveyand T. George Truscott (1999) Photophysical properties of5,10,15,20-tetrakis(m-hydroxyphenyl)porphyrin (m-THPP), 5,10,15,20-tetrakis(m-hydroxyphenyl)chlorin (m-THPC) and 5,10,15,20-tetra-kis(m-hydroxyphenyl)bacteriochlorin (m-THPBC): A comparativestudy. J. Chem. Soc. Perkin Trans. 2, 325–328.

39. Laville, I., T. Figueiredo, B. Loock, S. Pigaglio, P. Maillard, D. S.Grierson, D. Carrez, A. Croisy and J. Blais (2003) Synthesis, cellularinternalization and photodynamic activity of glucoconjugated deriva-tives of tri and tetra(meta-hydroxyphenyl)chlorins. Bioorg. Med.Chem. 11, 1643–1652.

40. Silva, A. M. G., A. C. Tom�e, M. G. P. M. S. Neves, A. M. S. Silva,J. A. S. Cavaleiro, D. Perrone and A. Dondoni (2002) Porphyrins in1,3-dipolar cycloaddition reactions with sugar nitrones. Synthesis ofglycoconjugated isoxazolidine-fused chlorins and bacteriochlorins.Tetrahedron. Lett. 43, 603–605.

41. Huang, Y.-Y., P. Mroz, T. Zhiyentayev, S. K. Sharma, T. Balasubra-manian, C. Ruzi�e, M. Krayer, D. Fan, K. E. Borbas, E. Yang, H. L.Kee, C. Kirmaier, J. R. Diers, D. F. Bocian, D. Holten, J. S. Lindseyand M. R. Hamblin (2010) In vitro photodynamic therapy and quan-titative structure�activity relationship studies with stable syntheticnear-infrared-absorbing bacteriochlorin photosensitizers. J. Med.Chem. 53, 4018–4027.

42. McCarthy, J. R., J. Bhaumik, N. Merbouh and R. Weissleder (2009)High-yielding syntheses of hydrophilic conjugatable chlorins andbacteriochlorins. Org. Biomol. Chem. 7, 3430–3436.

43. Yang, E., C. Ruzi�e, M. Krayer, J. R. Diers, D. M. Niedzwiedzki, C.Kirmaier, J. S. Lindsey, D. F. Bocian and D. Holten (2013) Photo-physical properties and electronic structure of bacteriochlorin–chal-cones with extended near-infrared absorption. Photochem. Photobiol.89, 586–604.

44. Silva, J. N., A. M. G. Silva, J. P. Tome, A. O. Ribeiro, M. R. M.Domingues, J. A. S. Cavaleiro, A. M. S. Silva, M. Graca, M. Neves,A. C. Tome, O. A. Serra, F. Bosca, P. Filipe, R. Santuse and P.Morliere (2008) Photophysical properties of a photocytotoxic fluori-nated chlorin conjugated to four b-cyclodextrins. Photochem. Photo-biol. Sci. 7, 834–843.

45. Drobizhev, M., A. Karotki, M. Kruk and A. Rebane (2002) Reso-nance enhancement of two-photon absorption in porphyrins. Chem.Phys. Lett. 355, 175–182.

46. H€anninen, P., J. Soukka and J. T. Soini (2008) Two-photon excita-tion fluorescence bioassays. Ann. N.Y. Acad. Sci. 1130, 320–326.

47. Garcia, G., F. Hammerer, F. Poyer, S. Achelle, M.-P. Teulade-Fic-hou and P. Maillard (2013) Carbohydrate-conjugated porphyrindimers: Synthesis and photobiological evaluation for a potentialapplication in one-photon and two-photon photodynamic therapy.Bioorg. Med. Chem. 21, 153–165.

48. Dy, J., K. Ogawa, A. Satake, A. Ishizumi and Y. Kobuke (2007)Water-soluble self-assembled butadiyne-bridged bisporphyrin: Apotential two-photon-absorbing photosensitizer for photodynamictherapy. Chem. Eur. J. 13, 3491–3500.

49. Diaspro, A., G. Chirico and M. Collini (2005) Two-photon fluores-cence excitation and related techniques in biological microscopy.Quarterly Rev. Biophys. 38, 97–166.

50. Rubart, M. (2004) Two-photon microscopy of cells and tissue. Circ.Res. 95, 1154–1166.

51. Mir, Y., J. E. van Lier, J.-F. Allard, D. Morris and D. Houde (2009)Two-photon absorption cross section of excited phthalocyanines by afemtosecond Ti-sapphire laser. Photochem. Photobiol. Sci. 8, 391–395.

52. Drobizhev, M., F. Meng, A. Rebane, Y. Stepanenko, E. Nickel and C.W. Spangler (2006) Strong two-photon absorption in new asymmetri-cally substituted porphyrins: Interference between charge-transfer andintermediate-resonance pathways. J. Phys. Chem. B 110, 9802–9814.

53. Starkey, J. R., A. K. Rebane, M. A. Drobizhev, F. Meng, A. Gong,A. Elliott, K. McInnerney and C. W. Spangler (2008) New two-pho-ton activated photodynamic therapy sensitizers induce xenografttumor regressions after near-IR laser treatment through the body ofthe host mouse. Clin. Cancer Res. 14, 6564–6573.

54. Dichtel, W. R., J. M. Serin, C. Edder, J. M. J. Fr�echet, M. Matu-szewski, L.-S. Tan, T. Y. Ohulchanskyy and P. N. Prasad (2004)Singlet oxygen generation via two-photon excited FRET. J. Am.Chem. Soc. 126, 5380–5381.

55. Balaz, M., H. A. Collins, E. Dahlstedt and H. L. Anderson (2009)Synthesis of hydrophilic conjugated porphyrin dimers for one-photonand two-photon photodynamic therapy at NIR wavelengths. Org.Biomol. Chem. 7, 874–888.

56. Dahlstedt, E., H. A. Collins, M. Balaz, M. K. Kuimova, M. Khur-ana, B. C. Wilson, D. Phillips and H. L. Anderson (2009) One- andtwo-photon activated phototoxicity of conjugated porphyrin dimerswith high two-photon absorption cross sections. Org. Biomol. Chem.7, 897–904.

57. Kuimova, M. K., H. A. Collins, M. Balaz, E. Dahlstedt, J. A. Levitt,N. Sergent, K. Suhling, M. Drobizhev, N. S. Makarov, A. Rebane,H. L. Anderson and D. Phillips (2009) Photophysical properties andintracellular imaging of water-soluble porphyrin dimers for two-pho-ton excited photodynamic therapy. Org. Biomol. Chem. 7, 889–896.

58. Drobizhev, M., Y. Stepanenko, A. Rebane, C. J. Wilson, T. E. O.Screen and H. L. Anderson (2006) Strong cooperative enhancementof two-photon absorption in double-strand conjugated porphyrin lad-der arrays. J. Am. Chem. Soc. 128, 12432–12433.

59. Easwaramoorthi, S., S. Y. Jang, Z. S. Yoon, J. M. Lim, C.-W. Lee,C.-L. Mai, Y.-C. Liu, C.-Y. Yeh, J. Vura-Weis, M. R. Wasielewskiand D. Kim (2008) Structure�property relationship for two-photonabsorbing multiporphyrins: Supramolecular assembly of highly-con-jugated multiporphyrinic ladders and prisms. J. Phys. Chem. A 112,6563–6570.

Photochemistry and Photobiology, 2014, 90 429

60. Biswas, S., H.-Y. Ahn, M. V. Bondar and K. D. Belfield (2011)Two-photon absorption enhancement of polymer-templated porphy-rin-based J-aggregates. Langmuir 28, 1515–1522.

61. Collini, E., C. Ferrante and R. Bozio (2007) Influence of excitonicinteractions on the transient absorption and two-photon absorptionspectra of porphyrin J-aggregates in the NIR region. J. Phys. Chem.C 111, 18636–18645.

62. Achelle, S., P. Couleaud, P. Baldeck, M.-P. Teulade-Fichou and P.Maillard (2011) Carbohydrate–porphyrin conjugates with two-photonabsorption properties as potential photosensitizing agents for photo-dynamic therapy. Eur. J. Org. Chem. 2011, 1271–1279.

63. Hammerer, F., S. Achelle, P. Baldeck, P. Maillard and M.-P. Teu-lade-Fichou (2011) Influence of carbohydrate biological vectors onthe two-photon resonance of porphyrin oligomers. J. Phys. Chem. A115, 6503–6508.

64. Ogawa, K., H. Hasegawa, Y. Inaba, Y. Kobuke, H. Inouye, Y.Kanemitsu, E. Kohno, T. Hirano, S.-I. Ogura and I. Okura (2006)Water-soluble bis(imidazolylporphyrin) self-assemblies with largetwo-photon absorption cross sections as potential agents for photody-namic therapy. J. Med. Chem. 49, 2276–2283.

65. Fisher, J. A. N., K. Susumu, M. J. Therien and A. G. Yodh (2009)One- and two-photon absorption of highly conjugated multiporphyrinsystems in the two-photon Soret transition region. J. Chem. Phys.130, 134506–134508.

66. Samaroo, D., M. Vinodu, X. Chen and C. M. Drain (2007) Meso-tetra(pentafluorophenyl) porphyrin as an efficient platform for combi-natorial synthesis and the selection of new photodynamic therapeu-tics using a cancer cell line. J. Comb. Chem. 9, 998–1011.

67. Drain, C. M. and S. Singh (2010) Combinatorial libraries of porphy-rins: Chemistry and applications. In The Handbook of Porphyrin Sci-ence With Applications to Chemistry, Physics, Materials Science,Engineering, Biology and Medicine, Vol. 1 (Edited by K. Kadish, K.M. Smith and R. Guilard). World Scientific Publisher, Singapore.

68. Thompson, S., X. Chen, L. Hui, A. Toschi, D. A. Foster and C. M.Drain (2008) Low concentrations of a non-hydrolysable tetra-S-gly-cosylated porphyrin and low light induces apoptosis in human breastcancer cells via stress of the endoplasmic reticulum. Photochem.Photobiol. Sci. 7, 1415–1421.

69. Silva, A. M. G., A. C. Tom�e, M. G. P. M. S. Neves, A. M. S. Silvaand J. A. S. Cavaleiro (2005) 1,3-Dipolar cycloaddition reactions ofporphyrins with azomethine ylides. J. Org. Chem. 70, 2306–2314.

70. Seybold, P. G. and M. Gouterman (1969) Porphyrins: XIII: Fluores-cence spectra and quantum yields. J. Mol. Spectrosc. 31, 1–13.

71. Bonnett, R. and G. Mart�õnez (2001) Photobleaching of sensitisersused in photodynamic therapy. Tetrahedron 57, 9513–9547.

72. Khurana, M., H. A. Collins, A. Karotki, H. L. Anderson, D. T.Cramb and B. C. Wilson (2007) Quantitative in vitro demonstrationof two-photon photodynamic therapy using photofrin� and visu-dyne�. Photochem. Photobiol. 83, 1441–1448.

73. Xu, C. and W. W. Webb (1996) Measurement of two-photon excita-tion cross sections of molecular fluorophores with data from 690 to1050 nm. J. Opt. Soc. Am. B 13, 481–491.

74. Fischer, M. and J. Georges (1996) Fluorescence quantum yield ofrhodamine 6G in ethanol as a function of concentration using ther-mal lens spectrometry. Chem. Phys. Lett. 260, 115–118.

75. Grin, M. A., I. S. Lonin, A. I. Makarov, A. A. Lakhina, F. V. Touk-ach, V. V. Kachala, A. V. Orlova and A. F. Mironov (2008) Synthe-sis of chlorin-carbohydrate conjugates by ‘click chemistry’.Mendeleev Commun. 18, 135–137.

76. Gouterman, M. (1978) The Porphyrins, Vol. III, 1 (Edited by D.Dolphin). Academic Press, New York.

77. Muthiah, C., M. Taniguchi, H.-J. Kim, I. Schmidt, H. L. Kee, D.Holten, D. F. Bocian and J. S. Lindsey (2007) Synthesis and photo-physical characterization of porphyrin, chlorin and bacteriochlorinmolecules bearing tethers for surface attachment. Photochem. Photo-biol. 83, 1513–1528.

78. Gentemann, S., C. J. Medforth, T. P. Forsyth, D. J. Nurco, K. M.Smith, J. Fajer and D. Holten (1994) Photophysical properties ofconformationally distorted metal-free porphyrins. Investigation intothe deactivation mechanisms of the lowest excited singlet state. J.Am. Chem. Soc. 116, 7363–7368.

79. Cseleny�ak, A., E. Pankotai, A. Csord�as, L. Kiss and Z. Lacza (2010)Live-cell fluorescent imaging of membrane or mitochondrion transferbetween connected cells in culture. In Microscopy: Science, Technol-ogy, Applications and Education, Vol. 1 (Edited by A. M�endez-Vilasand J. D�õaz), pp. 764–771. Formatex, Badajoz, Spain.

80. Oulmi, D., P. Maillard, J.-L. Guerquin-Kern, C. Huel and M. Mo-menteau (1995) Glycoconjugated porphyrins. 3. Synthesis of flatamphiphilic mixed meso-(glycosylated aryl)arylporphyrins and mixedmeso-(glycosylated aryl)alkylporphyrins bearing some mono- anddisaccharide groups. J. Org. Chem. 60, 1554–1564.

81. Laville, I., S. Pigaglio, J.-C. Blais, F. Doz, B. Loock, P. Maillard,D. S. Grierson and J. Blais (2006) In vitro phototoxicity of glyco-conjugated porphyrins and chlorins in colorectal adenocarcinoma(HT29) and retinoblastoma (Y79) cell lines. J. Med. Chem. 49,2558–2567.

430 Amit Aggarwal et al.