although singlet oxygen [ o2 ( g ) ] is an effective toxin for inactivating bacteria , methods to generate it suffer from photosensitizer problems including solubilization , degradation , and bleaching . turbid solutions can also present problem because light can be blocked from reaching the sensitizer . because of these issues , there is a need to develop methods for killing bacteria without the physical contact of photosensitizer with the solution . figure 1 shows the three - phase apparatus that we used in this study for the delivery of o2 to the air / water interface of a bacterial solution . by virtue of how the apparatus works , the solution is devoid of any photosensitizers , where gas - phase singlet oxygen diffuses to the solution surface . by analogy , majima et al . carried out experiments using a sensitizing tio2 surface and a terrylenediimide oxygen acceptor adsorbed on another surface that was separated by over 1 mm , indicating the formation of a diffusible o2 species ( similar to the kautsky three - phase test of 80 years ago ) . that the apparatus in figure 1 leads to o2 at the air / water interface for e. coli inactivation is not surprising because its design is similar to that of an apparatus invented by midden . what is new and better ( we regard our innovation as an offshoot of the midden and majima systems ) is the unique function of surfactant 1 in e. coli inactivation by airborne o2 . our hypothesis was that a o2-active surfactant ( 1 ) would synergistically enhance bacterial inactivation . it has been found in the photodynamic inactivation of bacteria with biofilm dispersions of a 2-aminoimidazole - triazole conjugate , in photodynamic therapy ( pdt ) with drug additives such as carboplatin , and with the simultaneous reaction of nitric oxide or so3 , among other o2 topics . similar to surfactant 1 , there was a report on a 2,5-disubstituted furan surfactant with a cationic tetraalkylammonium headgroup that was oxidized by o2 to an endoperoxide in a liposome study , but the reaction was not examined for antibacterial activity . here we show that an o2-active surfactant can synergistically enhance microbe inactivation from airborne o2 through hydroperoxide formation . our work serves as a starting point where in - situ - generated surfactant hydroperoxides function as secondary toxins to pure o2 for enhanced bactericidal action . following the experimental section , our results will be presented in four parts : first , the rationale for the selection of surfactant 1 ; second , measured surfactant photoperoxide formation via airborne o2 ; third , measured e. coli killing by o2 with and without surfactant 1 ; and fourth , measured e. coli killing by o2 , followed by the addition of hydroperoxides 2 and 3 in the dark . ( a ) red 669 nm light is directed in from above to a glass plate whose bottom side is coated with aluminum(iii ) phthalocyanine chloride tetrasulfonic acid ( pc ) . ( b ) o2 is sensitized by excited pc sites on the plate where o2 traverses a 0.41.5 mm distance to reach the e. coli solution of 0.1 mm surfactant 1 , where ( c ) hydroperoxides 2 and 3 are produced . porous vycor glass ( corning 7930 ) was purchased from advanced glass and ceramics ( holden , ma ) . silicon phthalocyanine dichloride , aluminum(iii ) phthalocyanine chloride tetrasulfonic acid , 9-bromo-2-methylnon-2-ene , sodium sulfite , triphenyl phosphine ( pph3 ) , benzoic acid , dimethylsulfone , dmf , ch2cl2 , ethanol , d2o , and dmso - d6 were purchased from commercial suppliers and were used as received . filter corporation deionization system ( vineland , nj ) . nuclear magnetic resonance ( nmr ) data were recorded on a bruker avance 400 spectrometer operating at 400 mhz for h nmr and at 100.6 mhz for c nmr . fab - mass spectrometry data were collected on a jeol jms - hx110 spectrometer using a m - nitrobenzyl alcohol matrix , a 10 kv acceleration voltage , and a xe beam fab gun ( 6 kv ) on the ms-1 ion source . an olympus fluoview fv10i confocal fluorescence microscope was used to analyze stained e. coli and assess membrane permeability following singlet oxygen exposure . using a pasteur pipet , 50 l of methanol containing 8  10 m aluminum(iii ) phthalocyanine chloride tetrasulfonic acid ( pc ) was deposited onto one side of pvg ( disk shape 14.0 mm  1.0 mm or square shape 2.25 cm and 1.01.5 mm ) . most of the methanol had evaporated after 1224 h at 26 c , at which point the sample was used . the result was pvg sensitizing glass loaded on one side with 1.1  10 mols of pc / g of pvg with the penetration of the sensitizer into the glass core and edges . a three - phase apparatus was constructed for airborne o2 delivery to the air / water interface of a solution ( figures 2 ) . the sensitizing glass plate was placed sensitizer face down , above a short quartz cuvette ( 1.0  1.0  0.7 cm ) containing 0.60 ml of water ( from a micropipet , precision 0.005 ml ) and illuminated perpendicularly from a 3.0 cm distance with 669 nm light ( 383 mw ) from a diode laser ( model 7404 , intense , north brunswick , nj ) . the 669 nm light was passed through an ft-400-emt optical fiber ( thorlabs , newton nj ) , which produced a gaussian distribution of incident photons on the sensitizing glass plate ( total dose  1700 j / cm ) . the diameter of the laser spot on the sensitizer glass plate was 0.95 cm ( area = 0.71 cm ) . the sensitizing glass plate sat atop the short cuvette above the water interface by 0.4 mm situated at the sides of the cuvette . moving laterally from the cuvette side to the midpoint of the meniscus , the distance between the sensitizer plate and water was 1.5 mm . these distances were measured with a miniature ruler and a 10 magnifying glass with an uncertainty of 0.04 mm . water evaporation was negligible and did not measurably change the volume over the course of a 1 h experiment . the water temperature was increased by 3.5  0.3 c in 1 h , which slightly reduces the lifetime of singlet oxygen ( by 10 ns ) . an analysis of the water samples after photolysis indicated that no pc molecules had dislodged from the sensitizing glass nor had any relocated from the glass to the water . apparatus for generating airborne o2 where it travels a short distance to a solution containing surfactant 1 and e. coli . ( a ) a sensitizer glass plate covers but does not contact the water solution in the quartz cuvette . ( b ) red 669 nm light is directed in from above via an optical fiber connected to a diode laser . a piece of white paper was placed in front of the beam and moved downward to capture the approximate path of the beam contacting the sensitizer plate ( nikon digital camera settings : iso 100 , f20 , and 1/50 s flash burst , 3 s total exposure ) . yield 38 mg ( 70% ) , purity > 98% . a strecker reaction between 9-bromo-2-methylnon-2-ene ( 0.05 g , 0.22 mmol ) and na2so3 ( 0.057 g , 0.45 mmol ) took place in 4 ml of refluxing dmf water ( 1:1 ) under a nitrogen atmosphere in 12 h. after the mixture was cooled to room temperature , 2 ml of deionized h2o and 2 ml of ethanol were added in succession . the filtrate was partitioned with ch2cl2 ( 4  4 ml ) , and the ch2cl2 fraction was discarded . the aqueous fraction was evaporated to dryness , leaving an off - white solid product that was recrystallized in ethanol  water ( 8:2 ) . h nmr ( d2o , 400 mhz ) :  5.1 ( t , j = 14.8 hz , 1h ) , 2.8 ( t , j = 16 hz , 2h ) , 1.93 ( m , 2h ) , 1.68 ( m , 2h ) , 1.64 ( s , 1h ) , 1.56 ( s , 1h ) , 1.36 ( m , 2h ) , 1.29 ( m , 4h ) . c nmr ( d2o , 100.6 mhz ) :  133.2 , 125.2 , 51.1 , 28.8 , 27.9 , 27.6 , 27.1 , 24.8 , 24.0 , 16.9 . ir ( neat )  2967 , 2916 , 2851 , 1465 , 1453 , 1155 cm . hrms ( fab ) m / z calcd for [ c10h19o3sna2 ] , 265.0849 ; found for [ c10h19o3sna2 ] , 265.0854 . g / l in deionized h2o , and the critical micellar concentration ( cmc ) was 9.7 mm on the basis of an nmr titration method similar to that of zhao and fung ( figure s9 , supporting information ) . after the reaction of 1 with o2 , water was removed under a stream of dry n2 . the residue was partitioned with chloroform ( 10  1 ml ) . the ratio of 2 and 3 was determined by h nmr analysis of the 4.8 and 5.5 ppm protons and comparison with benzoic acid as an internal standard in dmso - d6 . hydroperoxides 2 and 3 were stable enough for characterization as a mixture , but they began to decompose after 1 to 2 days in d2o at 26 c . h nmr ( dmso - d6 , 400 mhz ) :  11.2 ( s , 1h ) , 10.8 ( s , 1h ) , 5.5 ( m , 2h ) , 4.8 ( s , 2h ) , 4.1 ( t , j = 14 hz , 1h ) . c nmr ( dmso - d6 , 100.6 mhz ) :  145.1 , 135.2 , 129.6 , 113.3 , 88.2 , 80.8 , 52.0 , 32.3 , 30.7 , 29.4 , 28.8 , 25.5 , 17.2 . control reactions showed that 669 nm irradiation of a piece of native pvg that had no pc coating did not yield 2 and 3 . the addition of pph3 to the postreaction mixture led to corresponding allylic alcohols that were also detected by nmr . bacterial strain cw 3747 , a mutant of e. coli k12 , was purchased from the american type cells collection ( atcc ) . frozen stocks of e. coli ( 200 l ) were revived in 50 ml of luria broth ( lb ) for 1 h and diluted to amounts ranging from 15 to 50 g / ml as analyzed by uv  vis . water samples were exposed to airborne o2 via the apparatus for 10 min ( or 20 or 30 min up to 60 min , ) and 0.1 ml of the solution was poured onto agar plates . the samples were incubated at 37 c for 24 h to determine the quantity of active colonies . e. coli was treated with surfactant 1 ( 1 mm ) or hydroperoxides ( 0.010.2 mm 4:1 ratio of 2/3 ) at different time intervals of 2 min to 1 h to determine the dark toxicity . samples were assayed for cell viability by serial dilution in lb media and then plated in triplicate on lb with 1.5% agar . a live / dead baclight bacterial viability kit ( molecular probes , eugene , or ) was used to stain the e. coli cells with syto-9 ( diluted to 3.34 m ) and propidium iodide ( diluted to 20 m ) . bacteria samples ( 50 g / ml ) were centrifuged for 5 min at 10  000 g , and the pellets were suspended in deionized water by incubating each dye for 15 min at 37 c , followed by analysis with fluorescence microscope . error arises from three main sources : ( 1 ) compound weighing ( 2% ) , ( 2 ) nmr peak integration ( 2 to 3% ) , and ( 3 ) bacterial colony counting , which was done manually at three different rotation angles per plate ( reproducible to within 2% ) . an appealing hypothesis for the amplifying o2 toxicity is the use of a surfactant that can readily form a hydroperoxide product . thus , we selected terminally branched - chain olefin sulfonate 1 with an eye toward the ease of formation of allylic hydroperoxides . trisubstituted olefins are much more reactive with o2 ( 20500-fold ) than are di- and monosubstituted olefins . for example , the chemical quenching rate constant ( kr ) of o2 with 2-methyl-2-pentene is reasonably high ( 6  10 m s ) . in the case of the detergent concentration , we selected a relatively low 1 mm concentration of 1 so the hydrophobic group would preferably point away from the surface . our results show that the cmc of 1 ( c10h19so3 na ) ( 9.7 mm at 26 c ) is lower than that of straight - chain c10h21so3  na ( 43 mm at 25 c , but similar to that of straight - chain c12h25so3  na ( 9.8 or 12 mm at 25 c ) . although cmc s generally decrease for branched hydrophobic groups , this mainly applies to internal rather than terminal unsaturated sites of olefin sulfonates . by running experiments below the micellar concentrations of 1 , the surfactant tends not to aggregate into environments away from the air / water interface . the apparatus brought airborne o2 in from above onto the h2o or d2o solution for an ene reaction with 1 mm detergent 1 . the two hydroperoxides that were formed ( 2 and 3 ) have a shift of the double bond relative to that of 1 , which is a fingerprint reaction for singlet oxygen . we monitored the disappearance of 1 and the appearance of surfactant peroxides 2 and 3 ( mass balance 91% ) , where 2 and 3 were stable enough for characterization as a mixture but began to decompose after 1 to 2 days at 26 c . figure 3 shows that the airborne o2 oxidation of 1 led to hydroperoxide products ( 2 and 3 ) at double the efficiency in d2o as in h2o ( the yield was 18% in h2o and 33% in d2o ) . because o2 is not transferred deep into bulk water , it is not subject to the 20-fold  increase in d2o ( 69 s at 20 c ) as in h2o ( 3.5 s at 20 c ) . air moisture was found to introduce a small ( 0.2% ) amount of h2o into the d2o solution during the 1 h reaction period on the basis of the nmr integration of the hod signal , but this was not an explanation of the modest product increase in d2o . in d2o , airborne o2 reactions carried out with 1 above its cmc led a 2% yield of hydroperoxides 2 and 3 ( far less than the 33% yield with 1 below its cmc ) likely as a result of the micellar protection of the alkene site from incoming airborne o2 at the air / water interface . below the cmc , the results point to surface activity where airborne singlet oxygen attacks 1 in a partially solvated state . partially solvated 1 may relate to the observed stereoselectivity of hydroperoxides 2 and 3 because the ratio was 4:1 ( table 1 ) . thus , the hexyl sulfonate chain in 1 is not acting as a bulky allylic group as could have been expected from curling conformations or else 3 rather than 2 would be the major product . it is known that o2 geminal regioselectivity arises from bulky allylic groups on trisubstituted alkenes ( scheme 1 ) and with vinyl silanes . in the absence of sterics , the ene reaction of o2 with trisubstituted alkenes , such as 2-methyl-2-pentene , usually yields the secondary and tertiary hydroperoxides in an 1:1 ratio with a slight favoring of the secondary hydroperoxide in polar solvents . some control of hydroperoxide product selectivity has been found for photooxidations if the alkene is contained in nafion and zeolites . secondary and tertiary hydroperoxides have been seen to decompose at different rates when encapsulated within zeolites , but the obvious explanation that one of the hydroperoxides decomposes more rapidly prior to quantitation is not the case for 2 and 3 . we tentatively attribute the 4:1 ratio of 2/3 to o2 coming top down on the interface , where the methyl protons are surface  exposed  and more easily rotated than the methylene protons , with the latter being more wetted or anchored at the solution / air interface . in a transition - state model drawing ( scheme 2 ) , we propose that the distal oxygen of the perepoxide transition structure preferably abstracts a methyl hydrogen prior to surface h bonding . facile rotation may be key , where the methylene allylic hydrogens of the hexyl sulfonate chain are more restricted to rotation and thus less conformationally accessible ( higher barrier to rotation ) than the methyl groups . we do not think that electronic repulsion takes place between the distal perepoxide oxygen and the sulfonate anion to explain methyl rather than methylene h - abstraction regioselectivity . reaction of airborne singlet oxygen with surfactant 1 at the air / water interface in d2o (  , r = 0.9917 ) and in h2o (  , r = 0.9928 ) . airborne o2 is generated and crosses an intervening gap to the h2o or d2o solution of 1 ( 1.0 mm ) . ratio of product calculated from the integration of the h nmr 4.8 and 5.5 ppm signals . , we make a case that detergent 1 synergistically enhances the bactericidal action of incoming o2 . table 2 shows that the apparatus produces airborne o2 at levels toxic to bacteria ( entries 13 ) . samples containing 50 , 30 , and 15 g / ml e. coli were inactivated by 25 , 38 , and 41% , respectively , after 1 h. table 3 ( entry 5 ) shows that the inactivation of 50 g / ml e. coli when followed in 10 min increments led to 27% killing after 1 h. however , synergistic e. coli inactivation was seen when combining airborne o2 and surfactant 1 ( table 2 , entries 46 ) . that is , the number of e. coli killed increased by 1.7- to 2-fold compared to o2 treatment without 1 . the inactivation by 1 was 2.6% ( entry 7 ) and by airborne o2 was 25% ( entry 1 ) , which adds up to 27.6% , not the 50% seen with airborne o2 in the presence of surfactant 1 ( entry 4 ) . the synergism was not restricted to the 50 g / ml e. coli concentration but was also seen at 30 and 15 g / ml . table 2 shows that the surfactant 1 toxicity in the dark is low . for example , for 50 g / ml e. coli , 2.6% was killed by 1 mm 1 , and for 15 g / ml e. coli , 7.3% was killed ( entries 79 ) . the addition of a 4:1 mixture of 2 ( 0.144 mm ) and 3 ( 0.036 mm ) ( similar to the amount generated in situ in figure 3 ) in the dark was also relatively nontoxic , and the mixture led to 58% e. coli inactivation ( table 2 , entries 1012 ) . entry 13 shows a control reaction of the e. coli viability of 1.5% in the dark without surfactant 1 or hydroperoxides 2 and 3 . the red light emitted from the device was also mostly nontoxic to e. coli , and the inactivation ranged from 3.7 to 8% for e. coli concentrations of 50 to 15 g / ml ( table 2 , entries 1416 ) . these observations point to low levels of 38% e. coli inactivation based on additives 13 in the dark or in red light alone . next , we explored the effects of incubating hydroperoxides 2 and 3 with o2-pretreated cells . the above data suggest that airborne o2 with surfactant 1 enhanced singlet oxygen toxicity by an increase in oxidative stress ( e.g. , partial loss of cell membrane integrity ) . airborne o2 exposure was followed with the postreaction addition of a 4:1 mixture of 2 and 3 in the dark ( entries 15 ) . entries 15 ranged from 0.01 to 0.15 mm to mimic the hydroperoxide concentrations that form in situ for the reaction of 1 with airborne o2 in h2o in figure 3 . airborne o2 treatment for 1 h followed by the addition of 0.15 mm hydroperoxides 2 and 3 in the dark produced a similar inactivation of 50 g / ml e. coli ( 46% , table 3 , entry 5 ) compared to that of airborne o2 with surfactant 1 ( 50% , table 2 , entry 4 ) . the measured inactivation by hydroperoxides 2 and 3 was 5% , and by airborne o2 it was 25% , whereas the amount from the combination of airborne o2 and surfactant 1 was 50% , fully 20% greater inactivation . pre - exposure to airborne o2 with the postreaction addition of 1 in the dark did not enhance the e. coli inactivation ( table 3 , entry 6 ) . we believe that this enhanced inactivation is relevant to synergy , where o2-predamaged cells in the presence of 2 and 3 provoke cell killing . thus , we sought to gain insight into whether membrane damage was significant in o2-treated cells . we find evidence for cell permeabilization after o2 treatment in the presence or absence of 1 based on fluorescent labeling with a commercially available live / dead baclight bacterial viability kit ( figure s17 , supporting information ) . with syto-9 and propidium iodide stains added to 50 g / ml e. coli samples after treatment and centrifugation , the propidium iodide staining of cells indicated damaged membranes . consequently , we propose that airborne o2 causes permeabilization but that some cells can recover . however , the presence of hydroperoxides 2 and 3 may impede such a recovery by further destabilizing the cell . in a similar vein , redmond et al . attributes signaling and bystander effects to diffusing species such as h2o2 for the killing of neighboring cells adjacent to those photodynamically damaged . e. coli cells were treated with airborne o2 for 1 h. hydroperoxides 2 and 3 ( in a 4:1 ratio ) were added to the cells in the dark for 2 min . e. coli cells were treated with airborne o2 for 1 h. surfactant 1 was then added to the cells in the dark for 2 min . the results of this work show a heightened e. coli sensitivity to hydroperoxides produced in situ or added after airborne o2 treatment . we know that o2 exposure in the presence or absence of 1 leads to compromised cell membranes . we do not know the relative toxicities of 2 and 3 , for example , whether one hydroperoxide will cause greater membrane damage after the initial o2 reaction . our work also does not resolve whether 1 interacts with the cell membrane of the bacterium by adsorption or intercalation of its hydrophobic chain , but we believe that such sorption processes play a minor role as a result of the submicellar requirement mentioned earlier for hydroperoxide 2 and 3 formation . our interest in a relatively low 1 mm detergent 1 concentration was to potentially aim the hydrophobic group toward the surface , rather than aggregated it into a micelle away from the air / water interface . it turns out that reactive species preceding hydroperoxide formation are not likely to contribute to the toxicity because intermediates in the o2 ene reaction are usually not thought to form . in the absence of e. coli , we find no nmr evidence for facile hydroperoxide self - degradation , such as through hydroperoxide pair russell reactions , although we have not scrutinized hydroperoxide samples after 2 days when decomposition takes place . in summary , e. coli oxidation was carried out with airborne o2 and with the addition of 1 or hydroperoxides before and after o2 exposure to examine the mechanistic aspects . a majima  midden - like apparatus , as used here , exposes o2 to bacteria free from the effects of sensitizer pigmentation , bleaching , and degradation . here , the sensitizer glass plate was physically isolated from water as a means to inactivate bacteria . offering an innovative feature , the combination of airborne o2 with an oxidizable surfactant is promising . the arrival of airborne o2 to a water interface was used instead of its generation by a solvated photosensitizer . the apparatus has the advantage of being a source of gaseous singlet oxygen , otherwise the characteristics of surfactant solutions can change with added sensitizers and dyes . for airborne o2 bacterial killing , a future direction to develop may be in the presence of surfactants designed for rapid o2 oxygenation . . however , mono- and disubstituted alkenes are less reactive with o2 and thus less apt to amplify o2 toxicity . dark toxicity is also a concern , where anionic surfactants are often less so than cationic surfactants , such as quaternary ammonium surfactants , and would be advantageous . work on biofilms coated with active o2 ingredients or surfactants could be within reach of airborne o2 .
the reactivity of a trisubstituted alkene surfactant ( 8-methylnon-7-ene-1 sulfonate , 1 ) to airborne singlet oxygen in a solution containing e. coli was examined . 
 surfactant 1 was prepared by a strecker - type reaction of 9-bromo-2-methylnon-2-ene with sodium sulfite . 
 submicellar concentrations of 1 were used that reacted with singlet oxygen by an  ene 
 reaction to yield two hydroperoxides ( 7-hydroperoxy-8-methylnon-8-ene-1 sulfonate and ( e)-8-hydroperoxy-8-methylnon-6-ene-1 sulfonate ) in a 4:1 ratio . exchanging the h2o solution for d2o where the lifetime of solution - phase singlet oxygen increases by 20-fold led to an 2-fold increase in the yield of hydroperoxides pointing to surface activity of singlet oxygen with the surfactant in a partially solvated state . in this airborne singlet oxygen reaction , 
 e. coli inactivation was monitored in the presence and absence of 1 and by a live / dead cell permeabilization assay . 
 it was shown that the surfactant has low dark toxicity with respect to the bacteria , but in the presence of airborne singlet oxygen , it produces a synergistic enhancement of the bacterial inactivation . 
 how the ene - derived surfactant hydroperoxides can provoke 1o2 toxicity and be of general utility is discussed .