for materials and methods , see the supporting information . t. cruzi trans - sialidase,16c ,   which contained a hexahistidine tag , was expressed in e. coli m15 prep4 cells ( qiagen ) in terrific broth medium at 18 c for 16 h and 120 rpm .
the cells were harvested , re - suspended in 50 mm sodium phosphate buffer ( ph 8.0 ) that contained 0.3 m nacl and 0.05 % lubrol , and sonicated .
after centrifugation , the supernatant was applied onto a his - trap matrix ( ge healthcare ) and washed with 50 mm sodium phosphate buffer ( ph 8.0 ) that contained 0.3 m nacl .
trans - sialidase was eluted with 50 mm sodium phosphate buffer ( ph 8.0 ) that contained 250 mm imidazole with a purity of 95 % and dialyzed against 20 mm tris buffer ( ph 7.6 ) that contained 30 mm nacl .
sialyl lewis mimetics were tested for their ability to inhibit selectin - mediated binding by using a competitive spr assay , as described by enders et al .
briefly , recombinant selectin fc - chimeras ( r&d systems ) were coupled to gold nanoparticles that were coated with protein a ( 15 nm average , aurion ) in running buffer ( 20 mm hepes ph 7,4 ; 150 mm nacl , 1 mm cacl2 ) and passed over the surface of a sensor chip of a biacore x device ( ge healthcare ) .
the sensor - chip surface was divided into two flow cells : a measurement cell , which was functionalized with sle and sulfated tyrosine coupled to a polyacrylamide backbone , and a reference cell , which displays the non - binding lacnac polymer in a similar manner .
an aliquot ( 35 l ) of the selectin - coupled gold particles with the buffer or inhibitors was injected at a flow rate of 20 l min ; the dissociation phase was 120 s. the data points were collected by using the difference of the binding signal immediately before injection ( baseline ) and at the end of the dissociation phase .
the inhibitors were incubated for 18 min with the selectin gold particles prior to injection .
the resulting reduced binding signal was divided by the binding values without the inhibitor ( 100 % ) at each corresponding point , which was calculated by linear regression of the 100 % values taken before and after each series of measurements .
the ic50 values were calculated by using a dose - response curve fit ( log inhibitor versus normalized response - variable slope ) in graphpad prism 5 .
the title compound was prepared according to a method reported by neubacher et al.16b azide 8 ( 400 mg , 1.95 mmol ) and 4-nitrophenyl--sialoside 9 ( 492 mg , 1.10 mmol ) were dissolved in tris - hcl - buffer ( 8 ml , 100 mm , ph 7.9 ) .
the mixture was cooled to 13 c by using a cryostat and an aqueous solution of recombinant trans - sialidase ( 4 ml , c=0.93 mg ml ) was added .
the enzyme was denatured by the addition of etoh ( 10 ml ) and the mixture was centrifuged .
the lyophilisate was purified by flash chromatography on silica gel ( cyclohexane / etoac , 3:1etoac / meoh , 10:1meoh ) .
the product contained remaining tris - buffer ( 13 mol % as judged by nmr spectroscopy ) , which could neither be removed by the weakly acidic cation - exchange resin amberlite irc-86 nor by repeated chromatography .
yield : 257 mg ( 0.518 mmol , 47 % ) ; colorless solid ; rf=0.12 ( buoh / acoh / h2o , 5:2:2 ) ; m.p .
nmr ( cosy , 400 mhz , cd3od ) : =4.49 ( d , j1,2=8.5 hz , 1 h ; h-1 ) , 4.04 ( dd , j2,3=9.6 hz , j3,4=2.7 hz , 1 h ; h-3 ) , 3.95 ( d , j3,4=2.7 hz , 1 h ; h-4 ) , 3.863.59 ( m , 9 h ; h-5 , h-6a / b , h-4 , h-5 , h-6 , h-8 , h-9a / b ) , 3.55 ( pseudo - t , j1,2=j2,3=9.2 hz , 1 h ; h-2 ) , 3.50 ( m , 1 h ; h-7 ) , 2.84 ( dd , j=11.8 hz , j3,4=3.3 hz , 1 h ; h-3a ) , 2.01 ( s , 3 h ; ch3 ) , 1.761.70 ppm ( m , 1 h ; h-3b ) ; c nmr , hsqc ( 100.6 mhz , cd3od ) : =175.5 ( 2 c ; c = o , c = o ) , 101.1 ( c-2 ) , 92.3 ( c-1 ) , 77.7 ( c-3 ) , 70.3 ( c-2 ) , 70.0 ( c-7 ) , 78.9 , 74.9 , 72.9 , 69.3 ( c-5 , c-4 , c-6 , c-8 ) , 68.9 ( c-4 ) , 64.4 ( c-6 ) , 62.7 ( c-9 ) , 53.9 ( c-5 ) , 42.0 ( c-3 ) , 22.6 ppm ( ch3 ) ; ms ( esi ) : m / z ( % ) : 519.1 [ m+na ] ( 100 ) ; hrms ( esi ) : m / z calcd for [ c17h28n4o13+na ] : 519.1551 [ m+na ] ; found : 519.1546 .
fucosylacetylene 11 ( 82 mg , 0.19 mmol ) and azide 10 ( 91 mg , 0.18 mmol ) were dissolved in dry dmf ( 4 ml ) .
n , n - diisopropylethylamine ( dipea , 64 l ) and cui ( 7 mg , 0.04 mmol , 20 mol % ) were added in a countercurrent of argon gas and the mixture was stirred for 15 h at 70 c . the solvent was removed in vacuo and the residue was co - evaporated three times with toluene ( 5 ml each ) .
the crude product was purified by flash chromatography on silica gel ( ch2cl2/meoh , 4:12:1 ) to yield the title compound ( 94 mg , 0.10 mmol , 55 % yield ) as a light - yellow oil .
[ ]=51.6 ( c=0.6 , meoh ) ; h nmr ( cosy , 600 mhz , cd3od ) : =8.20 ( s , 1 h ; h-5 ) , 7.417.40 ( m , 2 h ; ph ) , 7.377.22 ( m , 13 h ; ph ) , 5.65 ( d , j1,2=8.9 hz , 1 h ; h-1 ) , 5.24 ( d , j1,2=5.4 hz , 1 h ; h-1 ) , 4.85 ( d , j=11.2 hz , 1 h ; ch2ph ) , 4.79 ( d , j=11.5 hz , 1 h ; ch2ph ) , 4.77 ( d , j=11.5 hz , 1 h ; ch2ph ) , 4.644.58 ( m , 3 h ; ch2ph ) , 4.27 ( dd , j2,3=9.6 hz , j3,4=2.0 hz , 1 h ; h-3 ) , 4.214.18 ( m , 2 h ; h-2 , h-2 ) , 4.09 ( d , j3,4=2.9 hz , 1 h ; h-4 ) , 4.06 ( dd , j2,3=8.9 hz , j3,4=2.9 hz , 1 h ; h-3 ) , 3.893.70 ( m , 9 h ; h-5 , h-6a , h-4 , h-5 , h-5 , h-6 , h-8 , h-9a / b ) , 3.633.60 ( m , 2 h ; h-6b , h-4 ) , 3.51 ( d , j=8.9 hz , 1 h ; h-7 ) , 2.90 ( dd , j=13.1 hz , j3,4=3.5 hz , 1 h ; h-3a ) , 2.01 ( s , 3 h ; ch3 ) , 1.801.79 ( m , 1 h ; h-3b ) , 1.19 ppm ( d , j5,ch3=6.4 hz , 3 h ; ch3 ) ; c nmr ( hsqc , hmbc , 150.9 mhz , cd3od ) : =175.5 ( 2 c ; c = o , c = o ) , 140.1 ( 3 c ; c-4 , 2cq  ph ) , 139.6 ( cq  ph ) , 129.4 ( 4 c ) , 129.34 ( 2 c ) , 129.27 ( 2 c ) , 129.2 ( 2 c ) , 129.0 ( 2 c ) , 128.79 , 128.77 , 128.67 ( ph ) , 124.4 ( 2 c ; c-5 , c-2 ) , 90.0 ( c-1 ) , 79.9 , 78.3 , 72.9 , 70.8 , 69.4 ( c-5 , c-4 , c-5 , c-6 , c-8 ) , 79.3 ( c-3 ) , 78.0 ( c-3 ) , 76.9 ( c-2 ) , 75.5 , 74.2 , 73.2 ( ch2ph ) , 75.0 ( c-4 ) ,
70.0 ( c-7 ) , 69.8 ( c-1 ) , 69.6 ( c-2 ) , 69.2 ( c-4 ) , 64.5 ( c-6 ) , 62.7 ( c-9 ) , 54.0 ( c-5 ) , 42.0 ( c-3 ) , 22.6 ( ch3 ) , 16.5 ppm ( ch3 ) ; ms ( esi ) : m / z ( % ) : 939.5 [ m+h ] ( 100 ) ; hrms ( esi ) : m / z calcd for [ c46h58n4o17+na ] : 961.3695 [ m+na ] ; found : 961.3692 .
triazole 12 ( 100 mg , 110 mol ) was dissolved in meoh ( 5 ml ) .
the solution was degassed by ultrasonication under an argon atmosphere before pd(oh)2 on charcoal ( 45 mg , 20 wt % ) was added . after a second degassing cycle ,
the argon atmosphere was replaced with hydrogen ( balloon ) . after stirring for 3 days at rt , another portion of the catalyst ( 45 mg ) was added , the hydrogen pressure was increased to 3.5 bar ( parr bomb ) , and the suspension was stirred for a further 5 days at rt .
the solvent was removed in vacuo and the crude product was purified by preparative hplc ( ace - c18-pfp ) to yield the title compound ( 24 mg , 36 mol , 33 % yield ) as a colorless oil .
= 39.1 ( c=0.5 , d2o ) ; h nmr ( cosy , noesy , roesy , 600 mhz , d2o ) : =8.36 ( s , 1 h ; h-5 ) , 5.78 ( d , j1,2=8.9 hz , 1 h ; h-1 ) , 5.30 ( d , j1,2=6.6 hz , 1 h ; h-1 ) , 4.32 ( dd , j2,3=9.7 hz , j3,4=3.0 hz , 1 h ; h-3 ) , 4.27 ( pseudo - t , j1,2=8.9 hz , j2,3=9.7 hz , 1 h ; h-2 ) , 4.19 ( dd , j1,2=6.6 hz , j2,3=10.4 hz , 1 h ; h-2 ) , 4.084.05 ( m , 2 h ; h-4 , h-3 ) , 3.98 ( t , j5,6=6.1 hz , 1 h ; h-5 ) , 3.893.80 ( m , 5 h ; h-4 , h-5 , h-5 , h-6 , h-9a ) , 3.73 ( d , j5,6=6.1 hz , 2 h ; h-6 ) , 3.683.66 ( m , 1 h ; h-4 ) , 3.613.58 ( m , 2 h , h-8 ; h-9b ) , 3.56 ( dd , j=9.1 hz , j=1.8 hz , 1 h ; h-7 ) , 2.73 ( dd , j=12.4 hz , j3,4=4.8 hz , 1 h ; h-3a ) , 1.99 ( s , 3 h ; ch3 ) , 1.80 ( pseudo - t , j=12.4 hz , j3,4=12.1 hz , 1 h ; h-3b ) , 1.14 ppm ( d , j5,ch3=6.5 hz , 3 h ; ch3 ) ; c nmr ( hsqc , hmbc , 150.9 mhz , d2o ) : =174.9 ( c = o ) , 173.8 ( c = o ) , 143.3 ( c-4 ) 124.8 ( c-5 ) , 99.9 ( c-2 ) , 87.6 ( c-1 ) , 78.0 ( c-5 ) , 75.7 ( c-3 ) , 72.8 ( c-8 ) , 71.7 ( c-6 ) , 71.6 ( c-4 ) , 70.5 ( c-1 ) , 70.1 ( c-3 ) , 69.3 ( c-5 ) , 68.3 ( c-4 ) , 68.0 ( c-7 ) , 67.9 ( c-2 ) , 67.6 ( c-4 ) , 67.2 ( c-2 ) , 62.5 ( c-9 ) , 60.8 ( c-6 ) , 51.6 ( c-5 ) , 39.4 ( c-3 ) , 22.0 ( ch3 ) , 15.6 ppm ( ch3 ) ; ms ( esi ) : m / z ( % ) : 691.3 [ m+na ] ( 100 ) , 669.3 [ m+h ] ( 89 ) ; hrms ( esi ) : m / z calcd for [ c25h40n4o17+na ] : 691.2286 [ m+na ] ; found : 691.2283 ; tr=2.1 min ( h2o / mecn , 98:298:2 ( 5 min)80:20 ( 15 min ) ; flow rate=38.00 ml min ) .
mannosylacetylene 14 ( 55.0 mg , 0.1 mmol ) and azide 10 ( 50.0 mg , 0.10 mmol ) were dissolved in dry dmf ( 2 ml ) .
dipea ( 35 l ) and cui ( 4.00 mg , 0.01 mmol , 10 mol % ) were added in a countercurrent of argon gas and the mixture was stirred for 15 h at 70 c .
the solvent was removed in vacuo and the residue was co - evaporated three times with toluene ( 5 ml each ) .
the crude product was purified by flash chromatography on silica gel ( ch2cl2/meoh , 2:0.2 + 0.1 % acohch2cl2/meoh 1:1 + 0.1 % acoh ) to yield the title compound ( 82.0 mg , 78.0 mol , 78 % yield ) as a light - yellow oil .
rf=0.57 ( ch2cl2/meoh 1:1 + 0.1 % acoh ) ; [ ]=+2.5 ( c=1.0 , meoh ) ; h nmr ( cosy , 400 mhz , cd3od ) : =8.16 ( s , 1 h ; h-5 ) , 7.397.37 ( m , 2 h ; ph ) , 7.337.22 ( m , 16 h ; ph ) , 7.187.15 ( m , 2 h ; ph ) , 5.66 ( d , j1,2=9.2 hz , 1 h ; h-1 ) , 5.30 ( d , j1,2=3.3 hz , 1 h ; h-1 ) , 4.75 ( d , j=11.0 hz , 1 h ; ch2ph ) , 4.70 ( d , j=12.3 hz , 1 h ; ch2ph ) , 4.69 ( d , j=11.6 hz , 1 h ; ch2ph ) , 4.61 ( d , j=11.6 hz , 1 h ; ch2ph ) , 4.594.52 ( m ,
2 h ; ch2ph ) , 4.514.49 ( m , 2 h ; ch2ph ) , 4.43 ( pseudo - t , j1,2=j2,3=3.3 hz , 1 h ; h-2 ) , 4.28 ( dd , j2,3=9.3 hz , j3,4=2.6 hz , 1 h ; h-3 ) , 4.22 ( pseudo - t , j1,2=j2,3=9.2 hz , 1 h ; h-2 ) , 4.09 ( d , j3,4=2.6 hz , 1 h ; h-4 ) , 3.99 ( pseudo - t , j3,4=j4,5=8.0 hz , 1 h ; h-4 ) , 3.95 ( dd , j2,3=3.3 hz , j3,4=8.0 hz , 1 h ; h-3 ) , 3.863.59 ( m , 12 h ; h-5 , h-6a / b , h-5 , h-6a / b , h-4 , h-5 , h-6 , h-8 , h-9a / b ) , 3.52 ( dd , j=8.9 hz , j=1.5 hz , 1 h ; h-7 ) , 2.89 ( dd , j=12.7 hz , j3,4=4.2 hz , 1 h ; h-3a ) , 2.03 ( s , 3 h ; ch3 ) , 1.821.76 ppm ( m , 1 h ; h-3b ) ; c nmr ( hsqc , 100.6 mhz , cd3od ) : =175.5 ( 2 c ; c = o , c = o ) , 146.2 ( c-4 ) , 139.8 , 139.7 , 139.6 , 139.5 ( 4cq  ph ) , 129.43 ( 2 c ) , 129.41 ( 4 c ) , 129.33 ( 2 c ) , 129.25 ( 2 c ) , 129.2 ( 2 c ) , 129.1 ( 2 c ) , 129.0 ( 2 c ) , 128.8 ( 2 c ) , 128.7 , 128.6 ( ph ) , 123.6 ( 2 c ; c-5 , c-2 ) , 90.0 ( c-1 ) , 79.9 ( c-3 ) , 77.9 ( c-3 ) , 76.6 ( c-2 ) , 76.0 ( c-4 ) , 75.3 , 74.3 , 73.3 , 73.1 ( ch2ph ) , 70.8 ( c-1 ) , 70.1 ( c-6 ) , 70.0 ( c-7 ) , 69.6 ( c-2 ) , 69.3 ( c-4 ) , 79.8 , 75.7 , 74.9 , 72.9 , 69.2 ( c-5 , c-5 , c-4 , c-6 , c-8 ) , 64.4 ( c-6 ) , 62.6 ( c-9 ) , 54.0 ( c-5 ) , 41.8 ( c-3 ) , 22.6 ppm ( ch3 ) ; ms ( esi ) : m / z ( % ) : 1045.5 [ m+h ] ( 70 ) ; hrms ( esi ) : m / z calcd for [ c53h65n4o18+h ] : 1045.4294 [ m+h ] ; found : 1045.4319 .
triazole 15 ( 155 mg , 148 mol ) was dissolved in meoh ( 7 ml ) and the solution was degassed by ultrasonication under an argon atmosphere .
then , pd on charcoal ( 50 mg , 10 wt % ) was added and , after a second degassing cycle , the argon atmosphere was replaced by hydrogen ( balloon ) . after stirring for 1 day at rt , no conversion was observed by tlc .
pd(oh)2 on charcoal ( 40 mg , 20 wt % ) was added and the mixture was stirred for 4 days at rt under a hydrogen atmosphere ; then , the catalyst was removed by filtration through celite and thoroughly washed with meoh .
the solvent was removed in vacuo and the crude product was purified by preparative hplc ( ace - c18-pfp ) to yield the title compound ( 63.0 mg , 92 mol , 62 % yield ) as a colorless oil .
[ ]=+5.4 ( c=1.25 , d2o ) ; h nmr ( cosy , 400 mhz , d2o ) : =8.33 ( s , 1 h ; h-5 ) , 5.80 ( d , j1,2=9.0 hz , 1 h ; h-1 ) , 5.24 ( d , j1,2=1.9 hz , 1 h ; h-1 ) , 4.554.54 ( m , 1 h ; h-2 ) , 4.33 ( dd , j2,3=9.6 hz , j3,4=2.9 hz , 1 h ; h-3 ) , 4.28 ( pseudo - t , j1,2=9.0 hz , j2,3=9.6 hz , 1 h ; h-2 ) , 4.09 ( d , j3,4=2.9 hz , 1 h ; h-4 ) , 4.00 ( t , j5,6=6.1 hz , 1 h ; h-5 ) , 3.883.81 ( m , 5 h ; h-6a / b , h-3 , h-5 , h9a ) , 3.783.73 ( m , 4 h ; h-4 , h-6a / b , h-8 ) , 3.703.66 ( m , 1 h ; h-4 ) , 3.633.57 ( m , 3 h ; h-5 , h-6 , h-9b ) , 3.443.41 ( m , 1 h ; h-7 ) , 2.75 ( dd , j=12.5 hz , j3,4=4.7 hz , 1 h ; h-3a ) , 2.01 ( s , 3 h ; ch3 ) , 1.831.79 ppm ( pseudo - t , j=12.5 hz , j3,4=12.1 hz , 1 h ; h-3b ) ; c nmr ( hsqc , 100.6 mhz , d2o ) : =175.0 ( c = o ) , 173.8 ( c = o ) , 144.1 ( c-4 ) , 123.6 ( c-5 ) , 100.0 ( c-2 ) , 87.7 ( c-1 ) , 78.0 ( c-5 ) , 75.7 ( c-3 ) , 75.4 ( c-7 ) , 72.8 ( 2 c ; c-1 , c-5 ) , 71.7 ( c-3 ) , 70.7 ( c-4 ) , 69.9 ( c-2 ) , 68.3 ( c-4 ) , 68.0 ( c-6 ) , 67.9 ( c-2 ) , 67.6 ( c-4 ) , 67.1 ( c-8 ) , 62.5 ( c-9 ) , 60.84 ( c-6 ) , 60.76 ( c-6 ) , 51.6 ( c-5 ) , 39.4 ( c-3 ) , 22.0 ppm ( ch3 ) ; ms ( esi ) : m / z ( % ) : 707.3 [ m+na ] ( 100 ) , 685.2 [ m+h ] ( 51 ) ; hrms ( esi ) : m / z calcd for [ c25h40n4o18+na ] : 707.2235 [ m+na ] ; found : 707.2232 ; tr=2.0 min ( h2o / mecn , 98:298:2 ( 5 min)80:20 ( 15 min ) ; flow rate=38.00 ml min ) .
to a solution of the peracetylated diglycosylindole 1911a ,   ( 26 mg , 33 mol ) in meoh ( 5 ml ) was added naome until the ph value reached 8.59.0 .
the mixture was stirred at rt for 18 h , neutralized with acoh , and the solvent was removed in vacuo .
the residue was purified by flash chromatography on silica gel ( etoac / meoh , 20:13:12:1 ) to yield the title compound ( 14 mg , 32 mol , 95 % yield ) as a colorless oil .
rf=0.45 ( 1-butanol / h2o / acoh , 5:2:2 ) ; [ ]=+61.0 ( c=1.00 , meoh ) ; h nmr ( cosy , hsqc , hmbc , 300 mhz , cd3od ) : =7.84 ( dd , j4,6=1.1 hz , j4,5=7.9 hz , 1 h ; h-4 ) , 7.58 ( dt , j7,5=0.8 hz , j7,6=8.4 , 1 h ; h-7 ) , 7.48 ( d , j2,1man=1.3 hz , 1 h ; h-2 ) , 7.18 ( ddd , j6,4=1.3 hz , j6,5=7.1 hz , j6,7=8.4 hz , 1 h ; h-6 ) , 7.08 ( ddd , j5,7=0.8 hz , j5,6=7.1 hz , j5,4=7.9 hz , 1 h ; h-5 ) , 5.41 ( d , j1,2=9.0 hz , 1 h ; h-1 ) , 5.34 ( pseudo - t , japp , 1,2-indol=1.3 hz , 1 h ; h-1 ) , 4.51 ( dd , j2,3=3.3 hz , j2,1=2.0 hz , 1 h ; h-2 ) , 4.22 ( pseudo - t , japp=9.3 hz , 1 h ; h-2 ) , 4.03 ( dd , j=3.2 , 0.9 hz , 1 h ; h-4 ) , 3.98 ( dd , j3,2=3.3 hz , j3,4=9.1 hz , 1 h ; h-3 ) , 3.693.86 ( m , 7 h ; h-3 , h-5 , h-6 , h-4 , h-6 ) , 3.26  ppm ( ddd , j5,6=2.9 hz , j5,6=4.0 hz , j5,4=9.4 hz , 1 h ; h-5 ) ; c nmr ( dept , hsqc , hmbc , 75 mhz , cd3od ) : =138.4 ( c-7a ) , 129.1 ( c-3a ) , 125.0 ( c-2 ) , 123.3 ( c-6 ) , 121.5 ( c-4 ) , 121.2 ( c-5 ) , 113.4 ( c-3 ) , 111.6 ( c-7 ) , 87.2 ( c-1 ) , 79.2 ( c-5 ) , 76.3 ( c-1 ) , 75.9 , 75.8 ( c-3 , c-5 ) , 73.5 ( c-3 ) , 72.1 ( c-2 ) , 71.1 ( c-2 ) , 70.5 ( c-4 ) , 69.3 ( c-4 ) , 62.8 , 62.6 ppm ( c-6 , c-6 ) ; ms ( esi ) : m / z ( % ) : 464.0 [ m+na ] ( 100 ) ; hrms ( esi ) : m / z calcd for [ c20h27no10+na ] : 464.1533 ; found : 464.1543 ;
m / z calcd for [ m+na+h ] : 464.1611 ; found : 464.1615 ; ir ( film ) : = 3271 , 2929 , 1705 , 1558 , 1461 , 1407 , 1272 , 1221 , 1064 , 1016 , 918 , 880 , 794 , 743 , 653 cm .
4-nitrophenyl--sialoside 9 ( 25 mg , 56 mol ) and diglycosylindol 20 ( 50 mg , 113 mol ) were dissolved in degassed tris - hcl buffer ( 2 ml , 100 mm , ph 7.5 ) .
after the addition of an aqueous solution of recombinant trans - sialidase ( 400 l , c=0.5 mg ml ) , the mixture was incubated at rt for 24 h. another portion ( 25 mg ) of compound 9 was added and the mixture was stirred for a further 48 h. the reaction was stopped by the addition of etoh ( 1.5 ml ) .
the crude product was purified by preparative hplc ( luna - c18 ; tr=2025 min ; h2o / mecn , 95:5 ( 60 min)80:20 ; flow rate=50 ml min ) to furnish the title compound ( 11 mg , 15 mol , 13 % yield ) as a yellowish solid .
the hplc separation also yielded unreacted acceptor 20 ( 22 mg , 50 mol ) , thus indicating that the yield of the enzymatic reaction was 24 % ( brsm ) .
[ ]=+81.6 ( c=1.00 , d2o ) ; h nmr ( cosy , tocsy , 600 mhz , d2o ) : =7.83 ( d , j4,5=8.0 hz , 1 h ; h-4 ) , 7.58 ( d , j7,6=8.4 hz , 1 h ; h-7 ) , 7.51 ( s , 1 h ; h-2 ) , 7.29 ( pseudo - t , j6,5/7=7.7 hz , 1 h ; h-6 ) , 7.18 ( pseudo - t , j5,4/6=7.6 hz , 1 h ; h-5 ) , 5.62 ( d , j1,2=8.4 hz , 1 h ; h-1 ) , 5.34 ( s , 1 h ; h-1 ) , 4.55 ( dd , j2,1=2.3 hz , j2,3=3.4 hz , 1 h ; h-2 ) , 4.274.32 ( m , 2 h ; h-2 , h-3 ) , 4.08 ( d , j4,3=2.6 hz , 1 h ; h-4 ) , 4.02 ( dd , j3,2=3.4 hz , j3,4=9.2 hz , 1 h ; h-3 ) , 3.943.97 ( m , 1 h ; h-5 ) , 3.84 ( ddd , j8,9a=2.5 hz , j8,9b=6.4 hz , j8,7=9.1 hz , 1 h ; h-8 ) , 3.783.81 ( m , 2 h ; h-5 , h-9a ) , 3.633.72 ( m , 6 h ; h-4 , h-6 , h-4 , h-6 ) , 3.61 ( dd , j6,7=1.6 hz , j6,5=10.5 hz , 1 h ; h-6 ) , 3.56 ( dd , j9b,8=6.4 hz , j9b,9a=12.0 hz , 1 h ; h-9b ) , 3.52 ( dd , j7,6=1.6 hz , j7,8=9.1 hz , 1 h ; h-7 ) , 3.23 ( ddd , j5,6=2.8 hz , j5,6=5.6 hz , j5,4=9.4 hz , 1 h ; h-5 ) , 2.72 ( dd , j3qu,2=4.6 hz , j3qu,3ax=12.3 hz , 1 h ; h-3qu ) , 1.96 ( s , 3 h ; coch3 ) , 1.80 ppm ( t , j3ax,3qu=12.3 hz , 1 h ; h-3ax ) ; c nmr ( hsqc , hmbc , 151 mhz , d2o ) : =174.9 ( c = o ) , 136.6 ( c-7a ) , 127.0 ( c-3a ) , 123.7 ( c-2 ) , 123.1 ( c-6 ) , 120.8 ( c-5 ) , 120.5 ( c-4 ) , 112.1 ( c-3 ) , 110.2 ( c-7 ) , 99.8 ( c-1 ) , 77.0 ( c-5 ) , 76.4 ( c-3 ) , 74.4 ( c-1 ) , 74.3 ( c-5 ) , 72.8 ( c-6 ) , 71.5 ( c-8 ) , 71.3 ( c-3 ) , 70.0 ( c-2 ) , 68.1 ( c-7 ) , 68.0 , 67.7 , 67.6 ( c-4 , c-4 , c-4 ) , 67.4 ( c-2 ) , 62.5 ( c-9 ) , 60.8 , 60.6 ( c-6 , c-6 ) , 51.5 ( c-5 ) , 39.3 ( c-3 ) , 21.9 ppm ( coch3 ) ; ms ( esi ) : m / z ( % ) : 755.3 [ m+na ] ( 100 ) ; hrms ( esi ) : m / z calcd for [ c31h44n2o18+na ] : 755.2487 ; found : 755.2514 .
t. cruzi trans - sialidase,16c ,   which contained a hexahistidine tag , was expressed in e. coli m15 prep4 cells ( qiagen ) in terrific broth medium at 18 c for 16 h and 120 rpm .
the cells were harvested , re - suspended in 50 mm sodium phosphate buffer ( ph 8.0 ) that contained 0.3 m nacl and 0.05 % lubrol , and sonicated .
after centrifugation , the supernatant was applied onto a his - trap matrix ( ge healthcare ) and washed with 50 mm sodium phosphate buffer ( ph 8.0 ) that contained 0.3 m nacl .
trans - sialidase was eluted with 50 mm sodium phosphate buffer ( ph 8.0 ) that contained 250 mm imidazole with a purity of 95 % and dialyzed against 20 mm tris buffer ( ph 7.6 ) that contained 30 mm nacl .
sialyl lewis mimetics were tested for their ability to inhibit selectin - mediated binding by using a competitive spr assay , as described by enders et al . briefly , recombinant selectin fc - chimeras ( r&d systems ) were coupled to gold nanoparticles that were coated with protein a ( 15 nm average , aurion ) in running buffer ( 20 mm hepes ph 7,4 ; 150 mm nacl , 1 mm cacl2 ) and passed over the surface of a sensor chip of a biacore x device ( ge healthcare ) .
the sensor - chip surface was divided into two flow cells : a measurement cell , which was functionalized with sle and sulfated tyrosine coupled to a polyacrylamide backbone , and a reference cell , which displays the non - binding lacnac polymer in a similar manner .
an aliquot ( 35 l ) of the selectin - coupled gold particles with the buffer or inhibitors was injected at a flow rate of 20 l min ; the dissociation phase was 120 s. the data points were collected by using the difference of the binding signal immediately before injection ( baseline ) and at the end of the dissociation phase .
the inhibitors were incubated for 18 min with the selectin gold particles prior to injection .
the resulting reduced binding signal was divided by the binding values without the inhibitor ( 100 % ) at each corresponding point , which was calculated by linear regression of the 100 % values taken before and after each series of measurements .
the ic50 values were calculated by using a dose - response curve fit ( log inhibitor versus normalized response - variable slope ) in graphpad prism 5 .
the title compound was prepared according to a method reported by neubacher et al.16b azide 8 ( 400 mg , 1.95 mmol ) and 4-nitrophenyl--sialoside 9 ( 492 mg , 1.10 mmol ) were dissolved in tris - hcl - buffer ( 8 ml , 100 mm , ph 7.9 ) . the mixture was cooled to 13 c by using a cryostat and an aqueous solution of recombinant trans - sialidase ( 4 ml , c=0.93 mg ml ) was added .
the enzyme was denatured by the addition of etoh ( 10 ml ) and the mixture was centrifuged .
the lyophilisate was purified by flash chromatography on silica gel ( cyclohexane / etoac , 3:1etoac / meoh , 10:1meoh ) .
the product contained remaining tris - buffer ( 13 mol % as judged by nmr spectroscopy ) , which could neither be removed by the weakly acidic cation - exchange resin amberlite irc-86 nor by repeated chromatography .
yield : 257 mg ( 0.518 mmol , 47 % ) ; colorless solid ; rf=0.12 ( buoh / acoh / h2o , 5:2:2 ) ; m.p .
175.0176.0 c ; [ ]=0.0 ( c=0.40 , cd3od ) ; h nmr ( cosy , 400 mhz , cd3od ) : =4.49 ( d , j1,2=8.5 hz , 1 h ; h-1 ) , 4.04 ( dd , j2,3=9.6 hz , j3,4=2.7 hz , 1 h ; h-3 ) , 3.95 ( d , j3,4=2.7 hz , 1 h ; h-4 ) , 3.863.59 ( m , 9 h ; h-5 , h-6a / b , h-4 , h-5 , h-6 , h-8 , h-9a / b ) , 3.55 ( pseudo - t , j1,2=j2,3=9.2 hz , 1 h ; h-2 ) , 3.50 ( m , 1 h ; h-7 ) , 2.84 ( dd , j=11.8 hz , j3,4=3.3 hz , 1 h ; h-3a ) , 2.01 ( s , 3 h ; ch3 ) , 1.761.70 ppm ( m , 1 h ; h-3b ) ; c nmr , hsqc ( 100.6 mhz , cd3od ) : =175.5 ( 2 c ; c = o , c = o ) , 101.1 ( c-2 ) , 92.3 ( c-1 ) , 77.7 ( c-3 ) , 70.3 ( c-2 ) , 70.0 ( c-7 ) , 78.9 , 74.9 , 72.9 , 69.3 ( c-5 , c-4 , c-6 , c-8 ) , 68.9 ( c-4 ) , 64.4 ( c-6 ) , 62.7 ( c-9 ) , 53.9 ( c-5 ) , 42.0 ( c-3 ) , 22.6 ppm ( ch3 ) ; ms ( esi ) : m / z ( % ) : 519.1 [ m+na ] ( 100 ) ; hrms ( esi ) : m / z calcd for [ c17h28n4o13+na ] : 519.1551 [ m+na ] ; found : 519.1546 .
fucosylacetylene 11 ( 82 mg , 0.19 mmol ) and azide 10 ( 91 mg , 0.18 mmol ) were dissolved in dry dmf ( 4 ml ) .
n , n - diisopropylethylamine ( dipea , 64 l ) and cui ( 7 mg , 0.04 mmol , 20 mol % ) were added in a countercurrent of argon gas and the mixture was stirred for 15 h at 70 c . the solvent was removed in vacuo and the residue was co - evaporated three times with toluene ( 5 ml each ) .
the crude product was purified by flash chromatography on silica gel ( ch2cl2/meoh , 4:12:1 ) to yield the title compound ( 94 mg , 0.10 mmol , 55 % yield ) as a light - yellow oil .
[ ]=51.6 ( c=0.6 , meoh ) ; h nmr ( cosy , 600 mhz , cd3od ) : =8.20 ( s , 1 h ; h-5 ) , 7.417.40 ( m , 2 h ; ph ) , 7.377.22 ( m , 13 h ; ph ) , 5.65 ( d , j1,2=8.9 hz , 1 h ; h-1 ) , 5.24 ( d , j1,2=5.4 hz , 1 h ; h-1 ) , 4.85 ( d , j=11.2 hz , 1 h ; ch2ph ) , 4.79 ( d , j=11.5 hz , 1 h ; ch2ph ) , 4.77 ( d , j=11.5 hz , 1 h ; ch2ph ) , 4.644.58 ( m , 3 h ; ch2ph ) , 4.27 ( dd , j2,3=9.6 hz , j3,4=2.0 hz , 1 h ; h-3 ) , 4.214.18 ( m , 2 h ; h-2 , h-2 ) , 4.09 ( d , j3,4=2.9 hz , 1 h ; h-4 ) , 4.06 ( dd , j2,3=8.9 hz , j3,4=2.9 hz , 1 h ; h-3 ) , 3.893.70 ( m , 9 h ; h-5 , h-6a , h-4 , h-5 , h-5 , h-6 , h-8 , h-9a / b ) , 3.633.60 ( m , 2 h ; h-6b , h-4 ) , 3.51 ( d , j=8.9 hz , 1 h ; h-7 ) , 2.90 ( dd , j=13.1 hz , j3,4=3.5 hz , 1 h ; h-3a ) , 2.01 ( s , 3 h ; ch3 ) , 1.801.79 ( m , 1 h ; h-3b ) , 1.19 ppm ( d , j5,ch3=6.4 hz , 3 h ; ch3 ) ; c nmr ( hsqc , hmbc , 150.9 mhz , cd3od ) : =175.5 ( 2 c ; c = o , c = o ) , 140.1 ( 3 c ; c-4 , 2cq  ph ) , 139.6 ( cq  ph ) , 129.4 ( 4 c ) , 129.34 ( 2 c ) , 129.27 ( 2 c ) , 129.2 ( 2 c ) , 129.0 ( 2 c ) , 128.79 , 128.77 , 128.67 ( ph ) , 124.4 ( 2 c ; c-5 , c-2 ) , 90.0 ( c-1 ) , 79.9 , 78.3 , 72.9 , 70.8 , 69.4 ( c-5 , c-4 , c-5 ,
c-6 , c-8 ) , 79.3 ( c-3 ) , 78.0 ( c-3 ) , 76.9 ( c-2 ) , 75.5 , 74.2 , 73.2 ( ch2ph ) , 75.0 ( c-4 ) , 70.0 ( c-7 ) , 69.8 ( c-1 ) , 69.6 ( c-2 ) , 69.2 ( c-4 ) , 64.5 ( c-6 ) , 62.7 ( c-9 ) , 54.0 ( c-5 ) , 42.0 ( c-3 ) , 22.6 ( ch3 ) , 16.5 ppm ( ch3 ) ; ms ( esi ) : m / z ( % ) : 939.5 [ m+h ] ( 100 ) ; hrms ( esi ) : m / z calcd for [ c46h58n4o17+na ] : 961.3695 [ m+na ] ; found : 961.3692 .
triazole 12 ( 100 mg , 110 mol ) was dissolved in meoh ( 5 ml ) .
the solution was degassed by ultrasonication under an argon atmosphere before pd(oh)2 on charcoal ( 45 mg , 20 wt % ) was added . after a second degassing cycle ,
the argon atmosphere was replaced with hydrogen ( balloon ) . after stirring for 3 days at rt ,
another portion of the catalyst ( 45 mg ) was added , the hydrogen pressure was increased to 3.5 bar ( parr bomb ) , and the suspension was stirred for a further 5 days at rt .
the solvent was removed in vacuo and the crude product was purified by preparative hplc ( ace - c18-pfp ) to yield the title compound ( 24 mg , 36 mol , 33 % yield ) as a colorless oil .
= 39.1 ( c=0.5 , d2o ) ; h nmr ( cosy , noesy , roesy , 600 mhz , d2o ) : =8.36 ( s , 1 h ; h-5 ) , 5.78 ( d , j1,2=8.9 hz , 1 h ; h-1 ) , 5.30 ( d , j1,2=6.6 hz , 1 h ; h-1 ) , 4.32 ( dd , j2,3=9.7 hz , j3,4=3.0 hz , 1 h ; h-3 ) , 4.27 ( pseudo - t , j1,2=8.9 hz , j2,3=9.7 hz , 1 h ; h-2 ) , 4.19 ( dd , j1,2=6.6 hz , j2,3=10.4 hz , 1 h ; h-2 ) , 4.084.05 ( m , 2 h ; h-4 , h-3 ) , 3.98 ( t , j5,6=6.1 hz , 1 h ; h-5 ) , 3.893.80 ( m , 5 h ; h-4 , h-5 , h-5 , h-6 , h-9a ) , 3.73 ( d , j5,6=6.1 hz , 2 h ; h-6 ) , 3.683.66 ( m , 1 h ; h-4 ) , 3.613.58 ( m , 2 h , h-8 ; h-9b ) , 3.56 ( dd , j=9.1 hz , j=1.8 hz , 1 h ; h-7 ) , 2.73 ( dd , j=12.4 hz , j3,4=4.8 hz , 1 h ; h-3a ) , 1.99 ( s , 3 h ; ch3 ) , 1.80 ( pseudo - t , j=12.4 hz , j3,4=12.1 hz , 1 h ; h-3b ) , 1.14 ppm ( d , j5,ch3=6.5 hz , 3 h ; ch3 ) ; c nmr ( hsqc , hmbc , 150.9 mhz , d2o ) : =174.9 ( c = o ) , 173.8 ( c = o ) , 143.3 ( c-4 ) 124.8 ( c-5 ) , 99.9 ( c-2 ) , 87.6 ( c-1 ) , 78.0 ( c-5 ) , 75.7 ( c-3 ) , 72.8 ( c-8 ) , 71.7 ( c-6 ) , 71.6 ( c-4 ) , 70.5 ( c-1 ) , 70.1 ( c-3 ) , 69.3 ( c-5 ) , 68.3 ( c-4 ) , 68.0 ( c-7 ) , 67.9 ( c-2 ) , 67.6 ( c-4 ) , 67.2 ( c-2 ) , 62.5 ( c-9 ) , 60.8 ( c-6 ) , 51.6 ( c-5 ) , 39.4 ( c-3 ) , 22.0 ( ch3 ) , 15.6 ppm ( ch3 ) ; ms ( esi ) : m / z ( % ) : 691.3 [ m+na ] ( 100 ) , 669.3 [ m+h ] ( 89 ) ; hrms ( esi ) : m / z calcd for [ c25h40n4o17+na ] : 691.2286 [ m+na ] ; found : 691.2283 ; tr=2.1 min ( h2o / mecn , 98:298:2 ( 5 min)80:20 ( 15 min ) ; flow rate=38.00 ml min ) .
mannosylacetylene 14 ( 55.0 mg , 0.1 mmol ) and azide 10 ( 50.0 mg , 0.10 mmol ) were dissolved in dry dmf ( 2 ml ) .
dipea ( 35 l ) and cui ( 4.00 mg , 0.01 mmol , 10 mol % ) were added in a countercurrent of argon gas and the mixture was stirred for 15 h at 70 c . the solvent was removed in vacuo and the residue was co - evaporated three times with toluene ( 5 ml each ) .
the crude product was purified by flash chromatography on silica gel ( ch2cl2/meoh , 2:0.2 + 0.1 %
acohch2cl2/meoh 1:1 + 0.1 % acoh ) to yield the title compound ( 82.0 mg , 78.0 mol , 78 % yield ) as a light - yellow oil .
rf=0.57 ( ch2cl2/meoh 1:1 + 0.1 % acoh ) ; [ ]=+2.5 ( c=1.0 , meoh ) ; h nmr ( cosy , 400 mhz , cd3od ) : =8.16 ( s , 1 h ; h-5 ) , 7.397.37 ( m , 2 h ; ph ) , 7.337.22 ( m , 16 h ; ph ) , 7.187.15 ( m , 2 h ; ph ) , 5.66 ( d , j1,2=9.2 hz , 1 h ; h-1 ) , 5.30 ( d , j1,2=3.3 hz , 1 h ; h-1 ) , 4.75 ( d , j=11.0 hz , 1 h ; ch2ph ) , 4.70 ( d , j=12.3 hz , 1 h ; ch2ph ) , 4.69 ( d , j=11.6 hz , 1 h ; ch2ph ) , 4.61 ( d , j=11.6 hz , 1 h ; ch2ph ) , 4.594.52 ( m , 2 h ; ch2ph ) , 4.514.49 ( m , 2 h ; ch2ph ) , 4.43 ( pseudo - t , j1,2=j2,3=3.3 hz , 1 h ; h-2 ) , 4.28 ( dd , j2,3=9.3 hz , j3,4=2.6 hz , 1 h ; h-3 ) , 4.22 ( pseudo - t , j1,2=j2,3=9.2 hz , 1 h ; h-2 ) , 4.09 ( d , j3,4=2.6 hz , 1 h ; h-4 ) , 3.99 ( pseudo - t , j3,4=j4,5=8.0 hz , 1 h ; h-4 ) , 3.95 ( dd , j2,3=3.3 hz , j3,4=8.0 hz , 1 h ; h-3 ) , 3.863.59 ( m , 12 h ; h-5 , h-6a / b , h-5 , h-6a / b , h-4 , h-5 , h-6 , h-8 , h-9a / b ) , 3.52 ( dd , j=8.9 hz , j=1.5 hz , 1 h ; h-7 ) , 2.89 ( dd , j=12.7 hz , j3,4=4.2 hz , 1 h ; h-3a ) , 2.03 ( s , 3 h ; ch3 ) , 1.821.76 ppm ( m , 1 h ; h-3b ) ; c nmr ( hsqc , 100.6 mhz , cd3od ) : =175.5 ( 2 c ; c = o , c = o ) , 146.2 ( c-4 ) , 139.8 , 139.7 , 139.6 , 139.5 ( 4cq  ph ) , 129.43 ( 2 c ) , 129.41 ( 4 c ) , 129.33 ( 2 c ) , 129.25 ( 2 c ) , 129.2 ( 2 c ) , 129.1 ( 2 c ) , 129.0 ( 2 c ) , 128.8 ( 2 c ) , 128.7 , 128.6 ( ph ) , 123.6 ( 2 c ; c-5 , c-2 ) , 90.0 ( c-1 ) , 79.9 ( c-3 ) , 77.9 ( c-3 ) , 76.6 ( c-2 ) , 76.0 ( c-4 ) , 75.3 , 74.3 , 73.3 , 73.1 ( ch2ph ) , 70.8 ( c-1 ) , 70.1 ( c-6 ) , 70.0 ( c-7 ) , 69.6 ( c-2 ) , 69.3 ( c-4 ) , 79.8 , 75.7 , 74.9 , 72.9 , 69.2 ( c-5 , c-5 , c-4 , c-6 , c-8 ) , 64.4 ( c-6 ) , 62.6 ( c-9 ) , 54.0 ( c-5 ) , 41.8 ( c-3 ) , 22.6 ppm ( ch3 ) ; ms ( esi ) : m / z ( % ) : 1045.5 [ m+h ] ( 70 ) ; hrms ( esi ) : m / z calcd for [ c53h65n4o18+h ] : 1045.4294 [ m+h ] ; found : 1045.4319 .
triazole 15 ( 155 mg , 148 mol ) was dissolved in meoh ( 7 ml ) and the solution was degassed by ultrasonication under an argon atmosphere .
then , pd on charcoal ( 50 mg , 10 wt % ) was added and , after a second degassing cycle , the argon atmosphere was replaced by hydrogen ( balloon ) . after stirring for 1 day at rt , no conversion was observed by tlc .
pd(oh)2 on charcoal ( 40 mg , 20 wt % ) was added and the mixture was stirred for 4 days at rt under a hydrogen atmosphere ; then , the catalyst was removed by filtration through celite and thoroughly washed with meoh .
the solvent was removed in vacuo and the crude product was purified by preparative hplc ( ace - c18-pfp ) to yield the title compound ( 63.0 mg , 92 mol , 62 % yield ) as a colorless oil .
[ ]=+5.4 ( c=1.25 , d2o ) ; h nmr ( cosy , 400 mhz , d2o ) : =8.33 ( s , 1 h ; h-5 ) , 5.80 ( d , j1,2=9.0 hz , 1 h ; h-1 ) , 5.24 ( d , j1,2=1.9 hz , 1 h ; h-1 ) , 4.554.54 ( m , 1 h ; h-2 ) , 4.33 ( dd , j2,3=9.6 hz , j3,4=2.9 hz , 1 h ; h-3 ) , 4.28 ( pseudo - t , j1,2=9.0 hz , j2,3=9.6 hz , 1 h ; h-2 ) , 4.09 ( d , j3,4=2.9 hz , 1 h ; h-4 ) , 4.00 ( t , j5,6=6.1 hz , 1 h ; h-5 ) , 3.883.81 ( m , 5 h ; h-6a / b , h-3 , h-5 , h9a ) , 3.783.73 ( m , 4 h ; h-4 , h-6a / b , h-8 ) , 3.703.66 ( m , 1 h ; h-4 ) , 3.633.57 ( m , 3 h ; h-5 , h-6 , h-9b ) , 3.443.41 ( m , 1 h ; h-7 ) , 2.75 ( dd , j=12.5 hz , j3,4=4.7 hz , 1 h ; h-3a ) , 2.01 ( s , 3 h ; ch3 ) , 1.831.79 ppm (
pseudo - t , j=12.5 hz , j3,4=12.1 hz , 1 h ; h-3b ) ; c nmr ( hsqc , 100.6 mhz , d2o ) : =175.0 ( c = o ) , 173.8 ( c = o ) , 144.1 ( c-4 ) , 123.6 ( c-5 ) , 100.0 ( c-2 ) , 87.7 ( c-1 ) , 78.0 ( c-5 ) , 75.7 ( c-3 ) , 75.4 ( c-7 ) , 72.8 ( 2 c ; c-1 , c-5 ) , 71.7 ( c-3 ) , 70.7 ( c-4 ) , 69.9 ( c-2 ) , 68.3 ( c-4 ) , 68.0 ( c-6 ) , 67.9 ( c-2 ) , 67.6 ( c-4 ) , 67.1 ( c-8 ) , 62.5 ( c-9 ) , 60.84 ( c-6 ) , 60.76 ( c-6 ) , 51.6 ( c-5 ) , 39.4 ( c-3 ) , 22.0 ppm ( ch3 ) ; ms ( esi ) : m / z ( % ) : 707.3 [ m+na ] ( 100 ) , 685.2 [ m+h ] ( 51 ) ; hrms ( esi ) : m / z calcd for [ c25h40n4o18+na ] : 707.2235 [ m+na ] ; found : 707.2232 ; tr=2.0 min ( h2o / mecn , 98:298:2 ( 5 min)80:20 ( 15 min ) ; flow rate=38.00 ml min ) .
to a solution of the peracetylated diglycosylindole 1911a ,   ( 26 mg , 33 mol ) in meoh ( 5 ml ) was added naome until the ph value reached 8.59.0 .
the mixture was stirred at rt for 18 h , neutralized with acoh , and the solvent was removed in vacuo .
the residue was purified by flash chromatography on silica gel ( etoac / meoh , 20:13:12:1 ) to yield the title compound ( 14 mg , 32 mol , 95 % yield ) as a colorless oil .
rf=0.45 ( 1-butanol / h2o / acoh , 5:2:2 ) ; [ ]=+61.0 ( c=1.00 , meoh ) ; h nmr ( cosy , hsqc , hmbc , 300 mhz , cd3od ) : =7.84 ( dd , j4,6=1.1 hz , j4,5=7.9 hz , 1 h ; h-4 ) , 7.58 ( dt , j7,5=0.8 hz , j7,6=8.4 , 1 h ; h-7 ) , 7.48 ( d , j2,1man=1.3 hz , 1 h ; h-2 ) , 7.18 ( ddd , j6,4=1.3 hz , j6,5=7.1 hz , j6,7=8.4 hz , 1 h ; h-6 ) , 7.08 ( ddd , j5,7=0.8 hz , j5,6=7.1 hz , j5,4=7.9 hz , 1 h ; h-5 ) , 5.41 ( d , j1,2=9.0 hz , 1 h ; h-1 ) , 5.34 ( pseudo - t , japp , 1,2-indol=1.3 hz , 1 h ; h-1 ) , 4.51 ( dd , j2,3=3.3 hz , j2,1=2.0 hz , 1 h ; h-2 ) , 4.22 ( pseudo - t , japp=9.3 hz , 1 h ; h-2 ) , 4.03 ( dd , j=3.2 , 0.9 hz , 1 h ; h-4 ) , 3.98 ( dd , j3,2=3.3 hz , j3,4=9.1 hz , 1 h ; h-3 ) , 3.693.86 ( m , 7 h ; h-3 , h-5 , h-6 , h-4 , h-6 ) , 3.26  ppm ( ddd , j5,6=2.9 hz , j5,6=4.0 hz , j5,4=9.4 hz , 1 h ; h-5 ) ; c nmr ( dept , hsqc , hmbc , 75 mhz , cd3od ) : =138.4 ( c-7a ) , 129.1 ( c-3a ) , 125.0 ( c-2 ) , 123.3 ( c-6 ) , 121.5 ( c-4 ) , 121.2 ( c-5 ) , 113.4 ( c-3 ) , 111.6 ( c-7 ) , 87.2 ( c-1 ) , 79.2 ( c-5 ) , 76.3 ( c-1 ) , 75.9 , 75.8 ( c-3 , c-5 ) , 73.5 ( c-3 ) , 72.1 ( c-2 ) , 71.1 ( c-2 ) , 70.5 ( c-4 ) , 69.3 ( c-4 ) , 62.8 , 62.6 ppm ( c-6 , c-6 ) ; ms ( esi ) : m / z ( % ) : 464.0 [ m+na ] ( 100 ) ; hrms ( esi ) : m / z calcd for [ c20h27no10+na ] : 464.1533 ; found : 464.1543 ; m / z calcd for [ m+na+h ] : 464.1611 ; found : 464.1615 ; ir ( film ) : = 3271 , 2929 , 1705 , 1558 , 1461 , 1407 , 1272 , 1221 , 1064 , 1016 , 918 , 880 , 794 , 743 , 653 cm .
4-nitrophenyl--sialoside 9 ( 25 mg , 56 mol ) and diglycosylindol 20 ( 50 mg , 113 mol ) were dissolved in degassed tris - hcl buffer ( 2 ml , 100 mm , ph 7.5 ) . after the addition of an aqueous solution of recombinant trans - sialidase
( 400 l , c=0.5 mg ml ) , the mixture was incubated at rt for 24 h. another portion ( 25 mg ) of compound 9 was added and the mixture was stirred for a further 48 h. the reaction was stopped by the addition of etoh ( 1.5 ml ) .
the crude product was purified by preparative hplc ( luna - c18 ; tr=2025 min ; h2o / mecn , 95:5 ( 60 min)80:20 ; flow rate=50 ml min ) to furnish the title compound ( 11 mg , 15 mol , 13 % yield ) as a yellowish solid .
the hplc separation also yielded unreacted acceptor 20 ( 22 mg , 50 mol ) , thus indicating that the yield of the enzymatic reaction was 24 % ( brsm ) .
[ ]=+81.6 ( c=1.00 , d2o ) ; h nmr ( cosy , tocsy , 600 mhz , d2o ) : =7.83 ( d , j4,5=8.0 hz , 1 h ; h-4 ) , 7.58 ( d , j7,6=8.4 hz , 1 h ; h-7 ) , 7.51 ( s , 1 h ; h-2 ) , 7.29 ( pseudo - t , j6,5/7=7.7 hz , 1 h ; h-6 ) , 7.18 ( pseudo - t , j5,4/6=7.6 hz , 1 h ; h-5 ) , 5.62 ( d , j1,2=8.4 hz , 1 h ; h-1 ) , 5.34 ( s , 1 h ; h-1 ) , 4.55 ( dd , j2,1=2.3 hz , j2,3=3.4 hz , 1 h ; h-2 ) , 4.274.32 ( m , 2 h ; h-2 , h-3 ) , 4.08 ( d , j4,3=2.6 hz , 1 h ; h-4 ) , 4.02 ( dd , j3,2=3.4 hz , j3,4=9.2 hz , 1 h ; h-3 ) , 3.943.97 ( m , 1 h ; h-5 ) , 3.84 ( ddd , j8,9a=2.5 hz , j8,9b=6.4 hz , j8,7=9.1 hz , 1 h ; h-8 ) , 3.783.81 ( m , 2 h ; h-5 , h-9a ) , 3.633.72 ( m , 6 h ; h-4 , h-6 , h-4 , h-6 ) , 3.61 ( dd , j6,7=1.6 hz , j6,5=10.5 hz , 1 h ; h-6 ) , 3.56 ( dd , j9b,8=6.4 hz , j9b,9a=12.0 hz , 1 h ; h-9b ) , 3.52 ( dd , j7,6=1.6 hz , j7,8=9.1 hz , 1 h ; h-7 ) , 3.23 ( ddd , j5,6=2.8 hz , j5,6=5.6 hz , j5,4=9.4 hz , 1 h ; h-5 ) , 2.72 ( dd , j3qu,2=4.6 hz , j3qu,3ax=12.3 hz , 1 h ; h-3qu ) , 1.96 ( s , 3 h ; coch3 ) , 1.80 ppm ( t , j3ax,3qu=12.3 hz , 1 h ; h-3ax ) ; c nmr ( hsqc , hmbc , 151 mhz , d2o ) : =174.9 ( c = o ) , 136.6 ( c-7a ) , 127.0 ( c-3a ) , 123.7 ( c-2 ) , 123.1 ( c-6 ) , 120.8 ( c-5 ) , 120.5 ( c-4 ) , 112.1 ( c-3 ) , 110.2 ( c-7 ) , 99.8 ( c-1 ) , 77.0 ( c-5 ) , 76.4 ( c-3 ) , 74.4 ( c-1 ) , 74.3 ( c-5 ) , 72.8 ( c-6 ) , 71.5 ( c-8 ) , 71.3 ( c-3 ) , 70.0 ( c-2 ) , 68.1 ( c-7 ) , 68.0 , 67.7 , 67.6 ( c-4 , c-4 , c-4 ) , 67.4 ( c-2 ) , 62.5 ( c-9 ) , 60.8 , 60.6 ( c-6 , c-6 ) , 51.5 ( c-5 ) , 39.3 ( c-3 ) , 21.9 ppm ( coch3 ) ; ms ( esi ) :
m / z ( % ) : 755.3 [ m+na ] ( 100 ) ; hrms ( esi ) : m / z calcd for [ c31h44n2o18+na ] : 755.2487 ; found : 755.2514 .
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 functional mimetics of the sialyl lewisx tetrasaccharide were prepared by the enzymatic sialylation of a 1,3-diglycosylated indole and a glycosyl azide , which was subsequently transformed into a 1,4-diglycosylated 1,2,3-triazole , by using the trans - sialidase of trypanosoma cruzi . 
 these compounds inhibited the binding of e- , l- , and p - selectin - coated nanoparticles to polyacrylamide - bound sialyl - lewisx - containing neighboring sulfated tyrosine residues ( styr / slex - paa ) at low or sub - millimolar concentrations . except for e - selectin 
 , the mimetics showed higher activities than the natural tetrasaccharide . 
Experimental Section
Expression and Purification of Trans-sialidase
Competitive Selectin-Inhibition Assay
3-O-(5-Acetamido-3,5-didesoxy-
1-(3-O-(5-Acetamido-3,5-didesoxy-
1-(3-O-(5-Acetamido-3,5-didesoxy-d-glycero--d-galactonon-2-ulopyranosyl))--d-galactopyranosyl-4-(-l-fucopyranosyl)-1 H-1,2,3-triazole (
1-(3-O-(5-Acetamido-3,5-didesoxy-
1-(3-O-(5-Acetamido-3,5-didesoxy-
1-(-
1-[3-O-(5-Acetamido-3,5-didesoxy-
Supporting Information