the total organic synthesis of 22-oh - pd1 ( 6 ) commenced 
 with an evans nagao acetate aldol reaction between the chiral auxiliary 8 and aldehyde 9 as previously reported ( scheme 1 ) . the alcohol 10 was formed in a 
 15.3:1 diastereomeric ratio when 8 was treated with ( i - pr)2net and ticl4 in ch2cl2 at 78 c followed by the addition of 
 freshly prepared aldehyde 9 . protection of the hydroxy 
 group in 10 was performed using standard conditions ( tbsotf , 
 2,6-lutidine ) , affording compound 11 in 84% yield over 
 the two steps . reductive removal of the auxiliary in 11 was achieved with dibal - h , yielding aldehyde 12 , which 
 was used directly in the next step ( scheme 1 ) . next , pent-4-ynoic acid ( 13 ) was converted 
 into the 
 wittig salt 14 in five steps and 42% overall yield as 
 previously reported . then aldehyde 12 was added to the ylide of 14 ; the latter formed 
 after reaction with nahmds in thf and hmpa at 78 c . stereochemically pure ester 15 was obtained in 47% yield 
 from 12 after purification by column chromatography ( scheme 1 ) . the alkyne 18 was prepared using a stereoselective 
 boron - mediated allylation reaction between 
 the chiral boron - auxiliary 16 and 3-(trimethylsilyl)propiolaldehyde 
 ( 17 ) using conditions reported by pietruszka and schne . removal of the tms group with meoh / k2co3 gave the terminal alkyne 19 , that was reacted 
 with the vinylic - bromide ester 15 in a sonogashira reaction 
 ( pd(pph3)4 , cui , et2nh ) . this yielded 
 ester 20 , containing all stereogenic centers and carbon 
 atoms present in 22-oh - pd1 ( 6 ) , in 65% yield over two 
 steps . removal of the tbs group went smoothly with excess tetra - n - butylammonium fluoride in thf to produce the triol - ester 21 in 86% yield . the boland protocol was employed in the reduction of the internal conjugated triple 
 bond in 21 , providing the methyl ester 7 in 40% yield after chromatography . in addition , < 1% of remaining 21 and approximately 23% of the over - reduced material 
 of 7 were obtained . all attempts to improve the yield 
 of 7 while retaining a high stereoselectivity were unsuccessful . 
 moreover , other methods explored did not give a stereoselective and 
 concurrent high - yielding reduction of alkyne 21 . the spectroscopic data ( uv , ir , nmr , and ms ) for the methyl ester 7 of 22-oh - pd1 ( 6 ) were in accord with the structure 
 ( supporting information ) . matching experiments using synthetic and biologically 
 produced 
 products were then conducted . first , the ester 7 was 
 hydrolyzed to the acid 6 under basic aqueous conditions . in order to determine whether synthetic material 
 matched biologically formed 6 , authentic 6 was obtained from human neutrophils . figure 1a shows the multiple reaction monitoring 
 ( mrm ) chromatograms of 22-oh - pd1 ( 6 ) from human neutrophils , 
 which gave a sharp peak at tr = 10.4 min . figure 1b depicts the mrm chromatogram of synthetic 
 22-oh - pd1 ( 6 ) , also with retention time tr = 10.4 min . figure 1c illustrates 
 the co - injection of synthetic and endogenous material added at essentially 
 equal amounts . overall , figure 1 demonstrates 
 that synthetic 6 coelutes with the material produced 
 by human neutrophils . prominent ions of diagnostic value were assigned 
 at m / z 375 = [ m  h ] , m / z 357 = [ m  
 h h2o ] , m / z 339 = [ m  h  2h2o ] , m / z 331 = [ m  h co2 ] , m / z 314 = [ m  h  2h2o  co2 ] , m / z 313 = 
 [ m  h  h2o co2 ] , and m / z 295 = [ m 
  h  co2  2h2o ] ( see the supporting information ) . the 
 uv and ms spectra for both natural and synthetic 6 were 
 essentially identical and in accord with the literature . n = 3 . selected 
 ion chromatogram ( m / z 375153 ) 
 depicting ( a ) 22-oh - pd1 ( 6 ) obtained from human peripheral 
 blood neutrophils ( 5  10/ml ) incubated with 0.1 
 mg of opsonized zymosan and pd1 ( 3 ) ( 10 ng / ml , 37 c , 
 30 min , dpbs , ph = 7.45 ) . ( b ) synthetic material ( inset : characteristic 
 uv - absorption spectrum in meoh , 1 nm ) . ( c ) co - injection of human 
 neutrophil 22-oh - pd1 ( 6 ) with synthetic 6 . during the acute phase of inflammation , 
 circulating polymorphonuclear 
 leukocytes ( pmn ) extravasate into the inflamed tissue . inhibition 
 of pmn infiltration and a decrease of pro - inflammatory mediators are 
 key processes in the resolution of inflammation and are defining actions 
 of spms . therefore , the anti - inflammatory 
 actions of 22-oh - pd1 ( 6 ) were investigated using an in 
 vivo zymosan - induced peritonitis mouse model . compound 6 was administrated 10 min prior to zymosan challenge , and peritoneal 
 inflammatory exudates were collected after 4 h. at 4 h 22-oh - pd1 ( 6 ) decreased pmn infiltration by 43% , to a similar 
 extent as pd1 ( 3 ) ( 54% ) itself ( figure 2 ) . synthetic material of pd1 ( 3 ) , matched 
 with biologically produced 3 , was employed . pd1 ( 3 ) and 22-oh - pd1 ( 6 ) limit 
 leukocyte 
 recruitment in vivo . mice were administered vehicle ( saline containing 
 0.1% etoh ) , pd1 ( 3 ) ( 10 ng / mouse ) , or 22-oh - pd1 ( 6 ) ( 10 ng / mouse ) i.p . 10 min prior to zymosan ( 1 mg / mouse , 
 i.p . ) . at 4 h peritoneal lavages . results for b are mean  sem ; n = 4 mice per treatment . * p < 0.05 , * * p < 0.01 versus zymosan - treated mice . because pro - inflammatory eicosanoid levels , including 
 ltb4 and pgf2 , increase during acute 
 inflammation , we next assessed the regulation 
 of local pro - inflammatory 
 eicosanoids by 22-oh - pd1 ( 6 ) using lc - ms / ms - based lipid 
 mediator metabololipidomics . mediators were identified in accordance 
 with published criteria that include 
 matching retention times ( figure 3a ) and ms - ms 
 fragmentation patterns ( figure 3b ) . quantification 
 of exudate lipid mediators by mrm demonstrated a significant reduction 
 in exudate eicosanoid levels including pgd2 ( 27% 
 decrease ) , pgf2 ( 43% decrease ) , and txb2 ( 48% decrease ; figure 3c ) , 
 following 22-oh - pd1 ( 6 ) administration , actions that 
 are also shared with pd1 ( 3 ) . together these results 
 demonstrate that 22-oh - pd1 ( 6 ) displays potent anti - inflammatory 
 actions in vivo . pd1 ( 3 ) and 22-oh - pd1 ( 6 ) reduce 
 local 
 pro - inflammatory eicosanoid levels in acute inflammation . lipid mediators 
 in peritoneal exudates collected 4 h after zymosan administration 
 ( 1 mg / mouse , i.p . ) were assessed using lc / ms - ms metabololipidomics 
 following solid - phase extraction . ( a ) representative mrm chromatograms 
 of selected ion pairs for arachidonic acid - derived eicosanoids , a 
 = 6-trans - ltb4 and b = 6-trans-12-epi - ltb4 . ( b ) representative ms - ms spectra with diagnostic 
 ions employed for the identification of ltb4 and pge2 . ( c ) quantification of exudate 
 lipid mediators following pd1 ( 3 ) and 22-oh - pd1 ( 6 ) administration ( 10 ng i.p . results are mean  sem ; n = 4 mice per group . 
 * p < 0.05 , * * p < 0.01 versus 
 zymosan - treated mice . spms regulate cell adhesion and transmigration to the site 
 of inflammation . as 22-oh - pd1 ( 6 ) displayed potent 
 actions on neutrophils in vivo , we next tested whether these findings 
 could be translated to humans by investigating the actions of synthetic 
 22-oh - pd1 ( 6 ) on human pmn chemotaxis and adhesion ( figure 4 ) . with primary human pmn , 22-oh - pd1 ( 6 ) was found to inhibit pmn chemotaxis to ltb4 ( 19% ) 
 and il-8 ( 23% ) ; see figure 4a and b , 
 respectively . compound 6 also inhibited pmn adhesion 
 to fibronectin under flow ( figure 4c ) . these 
 findings demonstrate that 22-oh - pd1 ( 6 ) displays potent 
 biological functions in vivo with mice and in vitro with human cells 
 that are comparable to its precursor pd1 ( 3 ) . pd1 ( 3 ) and 22-oh - pd1 ( 6 ) inhibit human 
 pmn chemotaxis and adhesion . purified human pmn were pretreated with 
 22-oh - pd1 ( 6 ) , pd1 ( 3 ) , or vehicle control 
 ( 15 min , 37 c ) . pmn were then cultured on a chemotx chamber 
 ( 90 min , 37 c in 5% co2 ) , and pmn chemotaxis toward 
 ( a ) ltb4 ( 10 nm ) or ( b ) il-8 ( 100 ng / ml ) was measured . ( c ) pmn were pretreated with 22-oh - pd1 ( 6 ) , pd1 ( 3 ) , or vehicle control ( 10 min , rt ) and stimulated 
 with 10 nm ltb4 ( 10 min , rt ) . pmn were then passed through 
 a flow chamber at 0.5 ml / min for 5 min . six random fields of view 
 were recorded , and the number of adhered pmn was quantified . representative 
 images of adhered pmn ( left panel ) and quantification ( right panel ) 
 are shown . results were obtained from three independent experiments , 
 shown as the mean  sem . all results were compared to vehicle 
 control using a paired t test ; statistical significance 
 defined as * p  0.05 , * * p  0.01 , * * * p  0.001 . in conclusion , a stereoselective synthesis of 
 the methyl ester 
 of 6 has been achieved in nine steps and 6% overall yield . furthermore , 22-oh - pd1 ( 6 ) was shown to maintain bioactive 
 properties resembling those of pd1 ( 3 ) , as it decreases 
 leukocyte recruitment in vivo , dampens production of classical pro - inflammatory 
 eicosanoids , and inhibits human pmn chemotaxis and adhesion . the -oxidation 
 of pd1 ( 3 ) to 22-oh - pd1 ( 6 ) is most likely , 
 as reported for some other spms , mediated by 
 cyp1 monooxygenases . in many cases -oxidation reactions reduce 
 the bioactivity of many local autacoids , i.e. , ltb4 . of 
 interest , the further metabolism of pd1 ( 3 ) gives a product , 
 namely , 22-oh - pd1 ( 6 ) , that retains the potent actions 
 of pd1 ( 3 ) . the results presented for 22-oh - pd1 ( 6 ) will be useful for future developments toward new pro - resolving 
 and anti - inflammatory drug candidates . optical rotations were 
 measured using a 1 ml cell with a 1.0 dm path length on a perkinelmer 
 341 polarimeter . the uv / vis spectra from 190 to 900 nm were recorded 
 using a biochrom libra s32pc spectrometer using quartz cuvettes . the 
 cd spectrum from 200 to 360 ir spectra ( 4000600 cm ) were obtained 
 on a perkinelmer spectrum bx series ft - ir spectrophotometer . nmr spectra 
 were recorded on a bruker avii400 spectrometer at 400 mhz for h nmr and at 101 mhz for c nmr . spectra are referenced 
 relative to the central residual protium solvent resonance in h nmr ( cdcl3  = 7.27 and meoh - d4  = 3.31 ) and the central carbon solvent resonance 
 in c nmr ( cdcl3  = 77.00 ppm and meoh - d4 =  49.00 ) . mass spectra were recorded 
 at 70 ev on a waters prospec q spectrometer using ei , es , or ci as 
 the method of ionization . high - resolution mass spectra were recorded 
 on a waters prospec q spectrometer using ei or es as the method of 
 ionization . thin - layer chromatography was performed on silica gel 
 60 f254 aluminum - backed plates fabricated by merck . flash column chromatography 
 was performed on silica gel 60 ( 4063 m ) produced by 
 merck . hplc analyses for chemical purities were performed on an agilent 
 technologies 1200 series instrument with diode array detector set 
 at 254 nm and equipped with a c18 stationary phase ( eclipse 
 xdb - c18 5 m 4.6  150 mm ) , applying the conditions 
 stated . unless stated otherwise , all commercially available reagents 
 and solvents were used in the form they were supplied without any 
 further purification . diastereomeric ratios reported in this paper have not been validated 
 by calibration ; see wernerova and hudlicky for discussions and guidelines . the eclipse plus c18 column 
 ( 100  4.6 mm  1.8 m ) was obtained from agilent , 
 and c18 spe columns were from waters . the synthetic standards 
 for lc - tandem mass spectrometry ( ms - ms ) quantitation and deuterated 
 ( d ) internal standards ( d4-pge2 and d4-ltb4 ) were purchased 
 from cayman chemicals . anti - mouse 
 cd11b and anti - mouse ly6 g were purchased from biolegend . compound 16 ( 275 mg , 0.50 mmol , 1.0 equiv ) was dissolved in ch2cl2 ( 0.25 ml ) and stirred at 0 c for 30 min 
 before tms - propargylaldehyde ( 17 ) ( 0.1 ml , 0.60 mmol , 
 1.2 equiv ) was added . the solution was stirred at 4 c for 24 
 h and another 4 days at rt before being concentrated in vacuo . the 
 crude product was purified by column chromatography on silica ( hexanes / etoac , 
 1:1 ) to afford the title compound 18 as a colorless oil . yield : 76 mg ( 72% ) ; [ ]d20 13 ( c 0.2 , meoh ) ; h nmr ( 400 mhz , cdcl3 )  5.695.54 
 ( m , 2h ) , 4.40 ( t , j = 5.9 hz , 1h ) , 3.64 ( ddd , j = 6.5 , 5.5 , 2.1 hz , 2h ) , 3.06 ( bs , 2h ) , 2.48 ( dd , j = 7.0 , 5.9 hz , 2h ) , 2.372.27 ( m , 2h ) , 0.15 ( s , 
 9h ) ; c nmr ( 101 mhz , cdcl3 )  129.9 , 
 127.2 , 106.5 , 89.3 , 61.9 , 61.7 , 35.5 , 30.7 , 0.0 ( 3c ) ; hrestofms m / z 235.1133 [ m + na ] ( calcd 
 for c11h20o2sina , 235.1130 ) ; tlc 
 ( hexanes / etoac , 1:1 , cam stain ) rf = 0.27 . the tms - alkyne ( 18 ) ( 130 mg , 
 0.61 mmol , 1.0 equiv ) was dissolved in dry meoh ( 20 ml ) , and k2co3 ( 80 mg ) was added . after 3 h , phosphate buffer 
 ( 20 ml , ph = 7.2 ) was added , and the aqueous phase was extracted with 
 etoac ( 4  30 ml ) . the combined organic layers were dried ( na2so4 ) , before being concentrated in vacuo . the crude 
 product was purified by column chromatography on silica ( hexanes / etoac , 
 4:6 ) to afford the title compound 19 as a yellow oil . yield : 81 mg ( 94% ) ; [ ]d20 9 ( c 0.2 , meoh ) ; h nmr ( 400 mhz , cdcl3 )  5.725.58 
 ( m , 2h ) , 4.464.41 ( m , 1h ) , 3.67 ( t , j = 5.9 
 hz , 2h ) , 3.46 ( bs , 1h ) , 2.60 ( bs , 1h ) , 2.552.49 ( m , 2h ) , 2.46 
 ( d , j = 2.2 hz , 1h ) , 2.382.31 ( m , 2h ) ; c nmr ( 151 mhz , cdcl3 )  130.3 , 126.9 , 84.6 , 
 73.0 , 61.7 , 61.4 , 35.4 , 30.7 ; hrestofms m / z 163.0739 [ m + na ] ( calcd for c8h12o2na , 163.0734 ) ; tlc ( hexanes / etoac , 4:6 , 
 cam stain ) rf = 0.17 . to a solution of vinyl bromide 15 ( 253 mg , 0.57 mmol , 1.0 equiv ) in et2nh ( 1.2 ml ) and 
 benzene ( 0.4 ml ) was added pd(pph3)4 ( 20 mg , 
 0.02 mmol , 3 mol % ) , and the reaction was stirred for 45 min in the 
 dark . cui ( 5 mg , 0.03 mmol , 5 mol % ) dissolved in a minimum amount 
 of et2nh was added , followed by dropwise addition of alkyne 19 ( 80 mg , 0.57 mmol , 1.0 equiv ) in et2nh ( 1.0 
 ml ) . after 20 h of stirring at ambient temperature , the reaction was 
 quenched by saturated nh4cl solution ( 15 ml ) . the aqueous phase 
 was extracted with et2o ( 2  15 ml ) , and the combined 
 organic layers were dried ( na2so4 ) , before being 
 concentrated in vacuo . the crude product was purified by column chromatography 
 on silica ( hexanes / etoac , 95:5 ) to afford the title compound 20 as a yellow oil . yield : 199 mg ( 69% ) ; [ ]d20 9 ( c 0.1 , meoh ) ; h nmr ( 400 mhz , cdcl3 )  6.56 ( dd , j = 15.6 , 10.9 hz , 1h ) , 6.19 
 ( dd , j = 15.3 , 11.0 hz , 1h ) , 5.78 ( dd , j = 15.1 , 5.9 hz , 1h ) , 5.735.62 ( m , 2h ) , 5.58 ( d , j = 15.6 hz , 1h ) , 5.465.32 ( m , 4h ) , 4.634.55 
 ( m , 1h ) , 4.20 ( q , j = 6.2 hz , 1h ) , 3.723.65 
 ( m , 5h ) , 2.832.73 ( m , 2h ) , 2.592.50 ( m , 2h ) , 2.422.34 
 ( m , 6h ) , 2.28 ( q , j = 6.9 , 6.1 hz , 2h ) , 0.89 ( s , 
 9h ) , 0.03 ( d , j = 9.1 hz , 6h ) ; c nmr 
 ( 151 mhz , cdcl3 )  173.7 , 141.9 , 139.8 , 130.1 , 129.9 , 
 129.4 , 128.5 , 128.0 , 127.2 , 125.7 , 110.1 , 92.0 , 84.3 , 72.7 , 62.3 , 
 62.0 , 51.7 , 36.4 , 35.7 , 34.2 , 30.8 , 26.0 ( 3c ) , 25.9 , 23.0 , 18.4 , 4.4 , 
 4.6 ; hrestofms m / z 525.3012 
 [ m + na ] ( calcd for c29h46o5sina , 525.3008 ) ; tlc ( hexanes / etoac , 1:1 , cam stain ) rf = 0.21 . tbaf ( 253 mg , 0.97 mmol , 2.5 equiv , 1.0 
 m in thf ) was added to a solution of tbs - protected alcohol 20 ( 195 mg , 0.39 mmol , 1.0 equiv ) in thf ( 5.5 ml ) at 0 c . the 
 reaction was stirred for 20 h before it was quenched with phosphate 
 buffer ( ph = 7.2 , 2.5 ml ) . brine ( 20 ml ) and etoac ( 20 ml ) were added , 
 and the phases were separated . the h2o phase was extracted 
 with etoac ( 2  20 ml ) , and the combined organic layer was dried 
 ( na2so4 ) before being concentrated in vacuo . the crude product was purified by column chromatography on silica 
 ( hexanes / etoac , 4:6 ) to afford the title compound 21 as 
 a pale yellow oil . yield : 129 mg ( 86% ) ; [ ]d20 25 ( c 0.1 , 
 meoh ) ; h nmr ( 400 mhz , cdcl3 )  6.56 
 ( dd , j = 15.5 , 10.9 hz , 1h ) , 5.83 ( dd , j = 15.2 , 6.0 hz , 1h ) , 5.745.58 ( m , 3h ) , 5.585.50 
 ( m , 1h ) , 5.445.32 ( m , 3h ) , 4.624.54 ( m , 1h ) , 4.24 
 ( q , j = 5.7 hz , 1h ) , 3.743.64 ( m , 5h ) , 2.902.72 
 ( m , 3h ) , 2.612.48 ( m , 2h ) , 2.422.30 ( m , 8h ) , 2.08 
 ( bs , 1h ) , 1.97 ( bs , 1h ) ; c nmr ( 101 mhz , cdcl3 )  173.8 , 141.5 , 138.3 , 131.6 , 130.1 , 129.4 , 129.1 , 128.2 , 
 127.2 , 124.8 , 110.9 , 92.4 , 84.2 , 71.6 , 62.3 , 61.9 , 51.8 , 35.6 , 35.4 , 
 34.1 , 30.8 , 25.9 , 23.0 ; hrestofms m / z 411.2147 [ m + na ] ( calcd for c23h32o5na , 411.2156 ) ; tlc ( hexanes / etoac , 4:6 , cam stain ) rf = 0.12 . alkyne 21 ( 25 
 mg , 0.064 mmol , 1.0 equiv ) was dissolved in h2o and meoh 
 ( 1:1 , 7.6 ml ) , to which a freshly prepared batch of zn(cu / ag ) ( boland 
 reagent ) ( 2.3 g ) was added in 
 one portion . the flask was evacuated and filled with argon ( 3 ) , 
 and the reaction mixture was stirred for 5 h. the suspension was vacuum 
 filtrated through a pad of celite , and the filter cake was washed 
 with additional fresh meoh . the filtrate was concentrated in vacuo , 
 to which brine and etoac were added . the phases were separated , and 
 the aqueous layer was extracted with etoac ( 3  10 ml ) . the organic 
 phase was dried ( na2so4 ) , filtrated , and concentrated 
 in vacuo . the crude product was purified by column chromatography 
 on silica ( heptane / etoac / meoh , 28:70:2 ) to afford the title compound 7 as a colorless oil . yield : 10 mg ( 40% ) ; [ ]d20 31 ( c 0.1 , meoh ) ; uv ( meoh ) max ( log  ) 
 262 ( 4.53 ) , 271 ( 4.60 ) , 282 ( 4.54 ) nm ; cd ( 0.1 g / ml , meoh ) , 
 max (  ) 343 ( 12.05 ) , 272 ( 15.09 ) , and 
 220 ( 6.20 ) nm ; ir ( neat ) max 3356 , 3014 , 2924 , 1734 , 
 1663 , 1436 , 1045 , 997 cm ; h nmr ( 400 
 mhz meoh - d4 )  5.305.18 
 ( m , 1h ) , 5.064.89 ( m , 2h ) , 4.79 ( t , j = 11.1 
 hz , 1h ) , 4.46 ( dd , j = 14.2 , 6.6 hz , 1h ) , 4.254.04 
 ( m , 7h ) , 3.353.23 ( m , 1h ) , 2.85 ( q , j = 6.5 
 hz , 1h ) , 2.36 ( s , 3h ) , 2.26 ( t , j = 6.8 hz , 2h ) , 
 1.591.44 ( m , 2h ) , 1.150.90 ( m , 10h ) ; c nmr ( 101 mhz , meoh - d4 )  175.3 , 
 138.0 , 135.0 , 134.8 , 131.4 , 130.9 , 130.6 , 130.3 , 129.1 , 129.0 , 128.9 , 
 128.1 , 126.5 , 73.0 , 68.5 , 62.6 , 52.1 , 36.6 , 36.4 , 34.8 , 32.0 , 26.7 , 
 23.8 ; hrestofms m / z 413.2505 [ m 
 + na ] ( calcd for c23h34o5na , 413.2298 ) ; tlc ( heptane / etoac / meoh , 28:70:2 , cam 
 stain ) rf = 0.30 . the 
 chemical purity ( > 96% ) was determined by hplc analysis ( eclipse 
 xdb - c18 , meoh / h2o , 65:35 , 1.0 ml / min ) : tr(minor ) = 7.52 and 8.02 min , and tr(major ) = 9.83 min . male fvb mice ( 6 to 8 weeks old ) 
 purchased from charles river laboratories were fed ad libitum laboratory 
 rodent diet 20 - 5058 ( lab diet , purina mills ) . animal experimental 
 procedures were approved by the standing committee on animals of harvard 
 medical school ( protocol no . zymosan ( 1 
 mg / ml ; sigma - aldrich ) was injected intraperitoneally ( i.p . ) 10 min 
 after i.p . administration of pd1 ( 3 ) or 22-oh - pd1 ( 6 ) ( 10 ng ) or vehicle ( 0.1% etoh in 100 ml of saline ) . at 
 4 h peritoneal lavages were collected in pbs without calcium and magnesium , 
 cells were enumerated as described by dalli et al . using light microscopy and flow cytometry , and local eicosanoid 
 levels were assessed by targeted lipid mediator metabololipidomics 
 as described below . all samples 
 for lc / ms - ms - based metabololipidomics were extracted with solid - phase 
 extraction columns as previously reported . prior to sample extraction , lavages were placed in two volumes of 
 ice - cold meoh containing d4-pge2 and d4-ltb4 , representing 
 each region in the chromatographic analysis ( 500 pg each ) to facilitate 
 quantification . extracted samples were analyzed by an lc / ms - ms system , 
 qtrap 6500 ( ab sciex ) equipped with a shimadzu sil-20ac autoinjector 
 and lc-20ad binary pump ( shimadzu corp . ) . an agilent eclipse plus 
 c18 column ( 100  4.6 mm  1.8 m ) was 
 used with a gradient of meoh / h2o / acetic acid of 55:45:0.01 
 ( vol : vol : vol ) that was ramped to 85:15:0.01 ( vol : vol : vol ) over 10 
 min and then to 98:2:0.01 ( vol : vol : vol ) for the next 8 min . this was 
 subsequently maintained at 98:2:0.01 ( vol : vol : vol ) for 2 min . the 
 flow rate was maintained at 0.4 ml / min . to monitor and quantify the 
 levels of pgd2 , pge2 , pgf2 , txb2 , and ltb4 , a multiple reaction monitoring 
 method was developed with signature ion fragments ( m / z ) for each molecule . identification was conducted 
 using published criteria where a minimum of six diagnostic ions were 
 employed . a linear calibration curve 
 for each compound was obtained with r values ranging from 0.98 to 0.99 . peripheral blood human 
 pmn were purified as previously described . in brief , using density - based ficoll - histopaque 1077 - 1 ( sigma ) , 
 pmn were isolated from human whole blood . purified pmn were resuspended in dpbs containing 
 0.1% bovine serum albumin and incubated with either vehicle ( < 0.03% 
 etoh by volume ) , pd1 ( 3 ) , or 22-oh - pd1 ( 6 ) ( 15 min , 37 c ) . for the chemotaxis assays , 1  10 pmn were plated on the upper chamber of a chemotx system 
 plate ( 3 m pore size filter , neuro probe ) , and chemotaxis toward 
 ltb4 ( 10 nm ) or il-8 ( 100 ng / ml , r&d systems ) was measured 
 after a 90 min incubation period ( 37 c in 5% co2 ) . the number of migrated pmn was measured using fluorescence - based prestoblue 
 quantification ( life technologies ) and read on a spectramax m3 plate 
 reader ( molecular devices inc . ) . purified human pmn were pretreated 
 for 10 min with 22-oh - pd1 ( 6 ) , pd1 ( 3 ) , 
 or vehicle control ; cells were then stimulated with 10 nm ltb4 . pmn ( 1  10 cells / ml ) were perfused over 
 fibronectin - coated ( r&d systems ) 1 -slide vi ibitreat microscopy chambers ( ibidi ) at 0.5 ml / min flow rate . six 
 fields of view were recorded per slide for 10 s. adherent pmn were 
 quantified using image proplus 7 ( mediacybernetics ) .
protectin d1 ( pd1 ( 3 ) ) , a c22-dihydroxylated polyunsaturated 
 fatty acid biosynthesized from all - z - docosahexaenoic 
 acid , belongs to the new family of endogenous mediators referred to 
 as specialized pro - resolving lipid mediators . 
 pd1 ( 3 ) 
 is a natural product that displays potent anti - inflammatory properties 
 together with pro - resolving actions including inhibition of polymorphonuclear 
 leukocyte ( pmn ) infiltration and promotion of macrophage phagocytosis 
 and efferocytosis . 
 given its potent endogenous actions , this compound 
 has entered several clinical development programs . 
 little has been 
 reported on the metabolism of pd1 ( 3 ) . 
 the synthesis 
 and biological evaluations of the -22 monohydroxylated metabolite 
 of pd1 ( 3 ) , named herein 22-oh - pd1 ( 6 ) , 
 are presented . 
 lc - ms / ms data of the free acid 6 , obtained 
 from hydrolysis of the synthetic methyl ester 7 , matched 
 data for the endogenously produced 22-oh - pd1 ( 6 ) . 
 compound 6 exhibited potent pro - resolving actions by inhibiting pmn 
 chemotaxis in vivo and in vitro comparable to its precursor pd1 ( 3 ) and decreased pro - inflammatory mediator levels in inflammatory 
 exudates . 
 the results reported herein provide new knowledge of the 
 metabolism of the protectin class of specialized pro - resolving mediators .