the bleomycins ( blm ) 
 are a family of glycopeptide - derived antitumor 
 antibiotics first isolated from streptomyces verticillus .
their antitumor activity derives from their ability 
 to effect dna cleavage in cancer cells and probably reflects the propensity of 
 the compounds to effect double - strand dna cleavage .
the bleomycins are also tumor - seeking 
 molecules , as demonstrated initially by the use of bleomycins derivatized 
 with radionuclides and more recently in cell culture experiments involving bleomycins 
 and blm carbohydrates conjugated to microbubbles and dyes .
bleomycin - mediated 
 dna strand scission requires oxygen and a metal 
 ion such as fe or cu , which activates blm for dna degradation .
the dna degradation process is initiated by the binding of activated 
 blm to its dna substrate ; the actual 
 chemistry of dna degradation has been characterized and shown to involve the rate - limiting abstraction 
 of a h atom from the c-4 position of a nucleoside deoxyribose 
 moiety .
as shown in figure 1 , the structure of bleomycin ( 1 ) includes a disaccharide 
 moiety composed of two unusual sugars , l - gulose and 3-o - carbamoyl - d - mannose .
interestingly , the disaccharide 
 moiety is not essential for dna cleavage in vitro , as the blm derivative lacking the disaccharide ( deglycoblm , 2 ) cleaves dna with a sequence selectivity and chemistry quite 
 similar to that of bleomycin itself , albeit with somewhat diminished 
 efficiency .
structures 
 of bleomycin a5 ( 1 ) , deglycobleomycin 
 a5 ( 2 ) , and bleomycin analogues 36 .
while the disaccharide moiety of blm is not essential for 
 dna cleavage , 
 we have recently demonstrated that the disaccharide is sufficient 
 to recapitulate the tumor cell targeting properties of bleomycin .
this suggested that bleomycin could potentially 
 be regarded as a modular system composed of a tumor - targeting agent 
 ( the disaccharide moiety ) and a tumoricidal agent ( deglycobleomycin ) . 

consistent with the need for the disaccharide moiety to effect efficient 
 cellular uptake in tumor cells , we find that deglycoblm is much less 
 cytotoxic than blm toward cultured cancer cells . to better define 
 the possible modular nature of bleomycin as an antitumor agent ,
we 
 have prepared derivatives of deglycoblm in which the blm disaccharide 
 is attached to the threonine moiety of deglycoblm ( blm 3 ) or to the c - terminal substituent ( blm 6 ) . also prepared 
 as negative controls were two blm derivatives related to 3 in which the disaccharide consisted of two anomers of l - gulose attached to 3-o - carbamoyl - d - glucose 
 or linked to deglycoblm through the threonine moiety ( blms 4 and 5 ) .
evaluation of the analogues for efficiency 
 of dna cleavage in a 
 plasmid dna relaxation assay revealed that blm 6 had 
 potency comparable to deglycoblm ( 2 ) , and that blms 35 were all significantly less potent . 

cytotoxicity testing was carried out in du145 human prostate cancer 
 cells . in spite of the weak dna cleavage activity of blm 3 ,
blms 4 and 5 , having disaccharides different than 
 the tumor - targeting disaccharide present in blm , were minimally cytotoxic . 

blm 6 , having the blm disaccharide attached at the c - terminus , 
 was found to be significantly more cytotoxic than deglycoblm in spite 
 of its comparable dna cleavage activity . to verify that the cytotoxicities 
 of the blm derivatives studied were strongly influenced by cellular 
 uptake , cytotoxic blms 1 , 2 , 3 , and 6 were conjugated to the cyanine dye cy5 * * ( affording 44 , 45 , 47 , and 55 , 
 respectively ) .
the dye - labeled compounds were shown to be internalized 
 by du145 cells , and the efficiency of uptake was in direct proportion 
 to the observed cytotoxicities .
these data underscore the importance 
 of the blm disaccharide for cellular uptake of this class of antitumor 
 agent and suggest that the cytotoxic activity of deglycoblm is limited 
 by lack of facile cellular uptake .
also studied in a preliminary 
 fashion was the possible basis for 
 cancer cell selective targeting and internalization by the carbohydrate 
 moiety of bleomycin .
evidence is presented that upregulation of one 
 or more receptors associated with enhanced glycolysis in tumor cells 
 may provide the means by which the bleomycins target tumor cells selectively .
bleomycin a5 was an outdated 
 clinical sample ; deglycobleomycin a5 was 
 obtained by treatment of bleomycin a5 with hf , followed 
 by purification on reversed - phase hplc .
the syntheses of bleomycin analogues 35 were accomplished by first preparing the requisite protected 
 glycosylated threonine derivatives 79 , respectively ( figure 2 ) .
structures of glycosylated 
 threonine derivatives 79 , the constituents 
 of bleomycin analogues 35 , respectively . synthesis of glycosylated threonine 
 derivative 7 began 
 with preparation of the protected 3-o - carbamoylated 
 mannose derivative 14 ( scheme 1 ) .
o - carbamoylated d - glucal 10(33 ) was treated with methyl trioxorhenium , urea hydrogen peroxide , 
 and dibutyl phosphate to afford the protected dibutyl phosphate derivatives 
 of 3-o - carbamoyl--d - glucopyranose 
 ( 11 ) and 3-o - carbamoyl--d - mannopyranose ( 12 ) in 30% and 27% yield , respectively .
the latter was o - acetylated to afford 13 as a colorless oil in 93% yield and then treated with thiophenol 
 and trimethylsilyl trifluoromethanesulfonate ( tmsotf ) , affording 14 , which crystallized as colorless needles in 92% yield .
conditions : ( a ) methyl trioxorhenium 
 ( 4 mol % ) , urea hydrogen peroxide , dibutyl phosphate , pyrazole , 4 
  molecular sieves , ch2cl2 ( 30% for 11 ; 27% for 12 ) .
( b ) ac2o , pyridine , 
 0 to 25 c ( 93% ) .
( c ) phsh , tmsotf , 4  molecular sieves , 
 ch2cl2 , 78 to 25 c ( 92% ) .
the perbenzylated orthoacetal of -l - gulopyranose 
 ( 15 ) was converted to 3,4,6-tri - o - benzyl-,-l - gulopyranosyl fluoride 
 ( 16 ) in 95% yield by treatment with diethylaminosulfur 
 trifluoride ( dast ) ( scheme 2 ) .
following o - deacetylation , 
 a solution of gulosyl fluoride 17 and phenylthiomannoside 14 in ch2cl2 was treated with n - iodosuccinimide and catalytic silver triflate at low temperature , affording disaccharide 18 in 91% yield as a colorless foam .
treatment of fluorinated 
 disaccharide 18 with fmoc - l - threonine benzyl 
 ester ( 19 ) in the presence 
 of cp2zrcl2 and agotf gave glycosylated threonine 
 derivative 20 as a colorless foam in 68% yield .
compound 20 was desilylated with 48% hf in water and then bisacetylated 
 with ac2o in pyridine .
finally , replacement of 
 the fluorenylmethyloxycarbonyl ( fmoc ) protecting group with a tert - butyloxycarbonyl ( boc ) protecting group , debenzylation with h2 over pd(oh)2/c , and reinstallation of the fmoc protecting group afforded 7 as a colorless solid in 57% overall yield .
key intermediate 7 was then converted to blm analogue 3 by solid - phase 
 synthesis in an overall 
 yield of 12% for the final four steps of the synthesis and hplc purification 
 ( scheme 3 ) .
conditions : ( a ) dast , ch2cl2 , 0 to 25 c ( 95% ) .
agotf , 4  molecular sieves , ch2cl2 , 78 
 to 5 c ( 91% ) .
( d ) cp2zrcl2 , agotf , 
 4  molecular sieves , ch2cl2 , 15 
 c ( 68% ) .
( h ) pd(oh)2/c , h2 , 1:1 
 thf  meoh ( 95% ) .
synthesis began from the protected dibutyl 
 phosphate derivative of 3-o - carbamoyl--d - glucopyranose ( 11 ) described above ( scheme 1 ) .
following acetylation , intermediate 28 was converted to the respective phenylthiopyranoside ( 29 ) in 89% yield by treatment with thiophenol and tmsotf ( scheme s1 , supporting information ) .
fmoc - l - threonine 
 benzyl ester ( 19 ) and gulopyranosyl fluoride 16 were treated with cp2zrcl2 and agotf , affording 
 -l - gulopyranosyl threonine derivative 30 as a colorless oil in 38% yield .
also formed in the same reaction 
 was -l - gulopyranosyl threonine derivative 37 ( 48% yield ) , which was employed for the synthesis of glycosylated 
 threonine 9 ( scheme s2 , supporting 
 information ) .
following deacetylation of 30(41 ) to afford 31 , treatment with 29 in the presence of n - iodosuccinimide and 
 agotf gave threonyl disaccharide 32 as a colorless foam in 70% yield .
desilylation ( 48% hf 
 in h2o , 96% yield ) afforded 33 , which was 
 peracetylated and converted to the respective boc derivative 35 in 89% overall yield .
hydrogenolysis over pd(oh)2/c and subsequent restoration of the fmoc protecting group afforded 8 in 65% overall yield from 35 .
intermediate 8 was then converted to blm analogue 4 , by analogy 
 with the synthesis of blm 3 from intermediate 7 ( cf .
scheme 3 ) . the strategy employed for synthesis 
 of glycosylated threonine derivative 9 ( scheme s2 , supporting information ) was closely analogous 
 to that used for the preparation of 8 .
-l - gulopyranosylthreonine derivative 37 , obtained in the 
 same reaction used for synthesis of 30 ( vide supra ) , 
 was deacetylated to afford 38 , the latter of which was 
 combined with phenylthiopyranoside 29 in the presence 
 of n - iodosuccinimde and 
 agotf , affording glycosylated threonine 39 as a colorless 
 foam in 63% yield .
desilylation and peracetylation gave 41 as a colorless foam in an overall yield of 72% .
replacement of the 
 fmoc protecting group with a boc group was effected in 68% yield , 
 and 42 was debenzylated by hydrogenolysis over pd(oh)2/c , providing 43 as a colorless solid in 97% 
 yield .
reinstallation of the fmoc protecting group then provided 9 in 70% overall yield from 43 .
intermediate 9 was converted to blm analogue 5 by analogy 
 with the synthesis of blm 3 from intermediate 7 ( cf scheme 3 ) .
synthesis of blm analogue 6 was 
 accomplished by use 
 of the peracetylated blm disaccharide 25 .
the anomeric 
 position of the gulose moiety was selectively deacetylated via the 
 agency of the acetate salt of hydrazine to afford 26 in 
 70% yield , and the disaccharide was then activated as the p - nitrophenylcarbonate derivative ( 27 ) ( scheme 4 ) .
condensation with the cu(ii ) salt of deglycoblm 
 a5 then afforded a carbamate linkage with the c - terminal 
 substituent , and subsequent deacetylation and demetalation provided 
 blm 6 in 39% yield .
( b ) p - nitrophenyl 
 chloroformate , 4-(n , n - dimethylamino)pyridine , 
 ch3cn ( 92% ) .
( c ) ( 1 ) cu(ii)deglycoblm a5 , dmf , net3 ; ( 2 ) nh2nh2 , dmf ; ( 3 ) 
 15% aqueous edta ( 39% ) .
shown in figure 3 is the cleavage of supercoiled 
 ( form i ) plasmid dna by blm derivatives 3 , 4 , and 5 in the presence of equimolar fe . while each of the analogues produced more form ii ( nicked plasmid ) 
 dna than fe alone when used at 4 m concentration , 

furthermore , no linear duplex ( form iii ) dna was apparent , indicating 
 an absence of double - strand dna cleavage . dna cleavage by blm 6 was also studied and the results are shown in figure 4 .
while fe(ii)blm 6 was less 
 potent at relaxing plasmid dna than fe(ii)blm a5 , 
 it produced slightly more form ii dna than fe(ii)deglycoblm 
 a5 and more form ii dna than the fe(ii ) chelates of blm 
 derivatives 3 , 4 , or 5 .
thus , 
 blm analogues 36 were all capable 
 of dna strand scission , but 6 was the most potent
. relaxation 
 of supercoiled pbr322 plasmid dna by blm a5 ( 1 ) and blm analogues 35 .
lane 1 , 
 dna alone ; lane 2 , 4 m fe ; lane 3 , 
 4 m blm a5 ( 1 ) ; lane 4 , 1 m 
 fe(ii)blm a5 ( 1 ) ; lane 5 , 4 m 
 fe(ii)blm 3 ; lane 6 , 2 m fe(ii)blm 3 ; lane 7 , 4 m fe(ii)blm 4 ; lane 
 8 , 2 m fe(ii)blm 4 ; lane 9 , 4 m fe(ii)blm 5 ; lane 10 , 2 m fe(ii)blm 5 .
the 
 numbers below each lane represent the percentage of cleaved dna relative 
 to all dna in the lane .
relaxation of supercoiled psp64 plasmid dna by blm a5 ( 1 ) and blm analogues 2 and 6 .
lane 
 1 , 2 m fe ; lane 2 , 4 m fe ; 
 lane 3 , 4 m blm a5 ( 1 ) ; lane 4 , 
 2 m fe(ii)blm a5 ( 1 ) ; lane 5 , 
 4 m fe(ii)blm a5 ( 1 ) ; lane 6 , 
 dna alone ; lane 7 , 2 m fe(ii)blm 6 ; lane 
 8 , 4 m fe(ii)blm 6 ; lane 9 , 2 m fe(ii)deglycoblm 
 ( 2 ) ; lane 10 , 4 m fe(ii)deglycoblm ( 2 ) .
the numbers below each lane represent the percentage of 
 cleaved dna relative to all dna in the lane .
blms 16 were tested for 
 their 
 cytotoxicity toward cultured du145 human prostate cancer cells ( figure 5 ) .
the cells were incubated for 24 h in the presence 
 of four different concentrations of each blm derivative .
cytotoxicity 
 was determined by use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 
 bromide ( mtt ) as a vital dye . as anticipated , blm a5 was 
 the most potent of the derivatives tested , presumably reflecting both 
 its efficient cleavage of dna ( figures 3 and 4 ) and the presence of the blm saccharide moiety , 
 which has been shown to enable tumor cell targeting and uptake .
deglycoblm a5 , lacking the tumor - targeting 
 disaccharide , was cytotoxic to du145 cells but much less so than blm 
 a5 .
blm 3 , which has the same disaccharide 
 moiety as blm itself , was slightly more cytotoxic to cultured du145 
 cells than deglycoblm , in spite of the fact that it is slightly less 
 potent in dna cleavage ( cf . figures 3 and 4 ) .
bleomycin derivatives 4 and 5 , which are isomeric with 3 but do not contain 
 the same disaccharide as blm , were only very weakly active as cytotoxins . 

compound 6 , containing the blm disaccharide attached 
 to the c - terminal substituent , was less cytotoxic than blm a5 but significantly more cytotoxic than deglycoblm or blm 3 .
thus , the presence of the blm disaccharide , attached to either 
 of two positions on deglycoblm other than the natural linkage via 
 -hydroxyhistidine , resulted in enhanced cytotoxicity .
the rank 
 order of cytotoxicities of blms 1 , 3 and 6 was the same as the order of their potencies of dna cleavage .
dose - dependent 
 effects of bleomycins 1 6 on the 
 viability of cultured du145 human prostate cancer 
 cells . to provide additional evidence 
 in support of the basis for expression 
 of cytotoxicity by glycosylated deglycoblms ,
the four cytotoxic derivatives 
 were conjugated to the cyanine dye cy5 * * ( figure 6 ) . as shown in figure 7 ,
the dye - labeled 
 conjugate of blm 1 ( 44 ) was internalized 
 the most efficiently , while uptake of dye - labeled deglycoblm 2 ( 45 ) was the least efficient .
the dye - labeled 
 conjugate of blm 6 ( 55 ) was internalized 
 more efficiently than the dye - labeled conjugate of blm 3 ( 47 ) but less efficiently than that of blm 1 ( 44 ) .
thus , the rank order of uptake of these conjugates 
 was precisely the same as the rank order of observed cytotoxicities 
 toward du145 cells .
structures of blm derivatives conjugated to cy5 * * , including 
 analogues 
 of blm 1 ( 44 ) , deglycoblm 2 ( 45 ) , blm 3 ( 47 ) , and blm 6 ( 55 ) .
( a ) comparison of the binding / internalization of dye - labeled analogues 
 of blm 1 ( 44 ) , deglycoblm 2 ( 45 ) , blm 3 ( 47 ) , and blm 6 ( 55 ) in du-145 prostate cancer cells and pz - hpv-7 
 normal prostate cells .
cells were treated with 25 m dye - labeled 
 conjugates at 37 c for 1 h , washed with phosphate - buffered saline 
 ( pbs ) , and fixed with 4% paraformaldehyde . the cell nuclei were stained 
 with diamidino-2-phenylindole ( dapi ) .
( b ) quantification of the binding / internalization 
 of dye - labeled conjugates in cultured prostate cancer and normal cells . in an effort to identify the cellular 
 receptor(s ) for blm and its 
 sugars
, we considered the possible involvement of metabolic events 
 characteristic of cancer cells , notably the shift to glycolysis for 
 atp production and away from mitochondrial oxidative phosphorylation . in an effort to mimic this process in normal 
 cells , normal lung wi-38 cells and normal kidney ccd-1105 kidtr cells 

were treated with a sublethal dose of the mitochondrial complex i 
 inhibitor rotenone for 24 h to force 
 the cells to use glycolysis to a greater extent . as shown in figure 8 , following rotenone treatment , both cell lines 
 exhibited the same enhanced update of blm - cy5 * * ( 44 ) 
 as did the numerous cancer cell lines we have studied .
effect of mitochondrial electron - transport chain ( etc ) 
 inhibitor 
 on internalization of the dye - labeled analogue of blm 1 ( 44 ) by cultured normal lung wi-38 cells and normal 
 kidney ccd-1105 kidtr cells .
cells were seeded to 16-well slides for 
 24 h , and then 25 m dye conjugate was added to the cells at 
 37 c for 1 h after the cells had been treated with 10 nm rotenone 
 for 24 h. cells were washed with pbs and fixed with 4% paraformaldehyde . 

the facilitative glucose transporter 
 glut1 is known to be inducible and is 
 subject to inhibition both by cytochalasin 
 b and phloretin , although neither of these compounds is specific for this receptor . 

as shown in figure 9 , both of these known glut1 
 inhibitors effected substantial and dose - dependent inhibition of blm - cy5 * * 
 binding / uptake in sw-480 colon cancer cells .
effect of glut1 inhibitors 
 on internalization of the dye - labeled 
 analogue of blm 1 ( 44 ) by sw-480 colon cancer 
 cells .
cells were pretreated with different concentrations of ( a ) 
 cytochalasin b or ( b ) phloretin at 37 c for 2 h before incubation 
 with 25 m dye - labeled conjugate and then washed with pbs and 
 fixed with 4% paraformaldehyde .
the present study was designed to define 
 the contributions of the 
 carbohydrate moiety of bleomycin to the dna - cleaving and cytotoxic 
 actions of this clinically used antitumor antibiotic .
deglycoblm , 
 the bleomycin analogue lacking the carbohydrate moiety , has been studied 
 fairly extensively and shown to cleave duplex dna by the same chemistry 
 as blm and with similar sequence selectivity .
however , the potency of supercoiled dna relaxation by deglycoblm 
 is 23-fold less than for blm , and much less double - strand 
 cleavage is apparent for deglycoblm .
two studies in which the disaccharide moiety was replaced with a 
 single sugar afforded blm analogues with diminished potency of dna 
 cleavage as well as a lesser ability to cleave rna .
thus , the disaccharide moiety attached to blm 
 via -hydroxyhistidine significantly potentiates dna cleavage 
 and enables double - strand dna cleavage and rna cleavage .
the 
 present study in du145 prostate cancer cells revealed that 
 deglycoblm was significantly less cytotoxic than blm .
it was initially 
 unclear whether this diminished cytotoxic potential of deglycoblm 
 was due to diminished potency of dna cleavage , lack of significant 
 double - strand cleavage , or lack of tumor cell targeting and uptake . 

the recent study from our laboratory documenting tumor targeting and 
 uptake by the disaccharide moiety itself underscores the ambiguity intrinsic to defining the role(s ) of the 
 blm disaccharide . in order to gain insights into possible roles 
 of the disaccharide
, 
 the present study considers the possibility that blm may be modular 
 in nature , composed of a carbohydrate moiety responsible for tumor 
 targeting and uptake and an aglycon that mediates dna strand scission . 

this hypothesis was tested by preparing analogues of blm in which 
 the disaccharide moiety is attached to blm through structural elements 
 other than the -hydroxyhistidine moiety .
if blm actually is 
 modular in nature , it might be anticipated that the disaccharide would 
 still target tumor cells and the aglycon would still exert a cytotoxic 
 effect via dna strand scission in spite of the altered relative orientations 
 of these two structural elements .
analogue 3 differs 
 from blm in that it has the normal 
 blm disaccharide attached through the threonine moiety .
this position 
 has been shown to be reasonably tolerant of substitution , although it has also been demonstrated that threonine is spatially 
 close to a structural element in blm that requires a specific conformation 
 for optimal expression of dna cleavage activity .
analogues 4 and 5 , containing disaccharides isomeric to 
 those in 3 , were anticipated to have dna cleavage activities 
 comparable to 3 but to lack any tumor targeting / uptake 
 properties .
finally , blm analogue 6 has a disaccharide 
 moiety identical with that of blm but attached to the end of the c - terminal 
 substituent by means of a carbamate linkage .
it was anticipated that 
 the disaccharide itself would have little effect on the efficiency 
 of dna cleavage , such that the potency of dna cleavage should be similar 
 to that of deglycoblm a5 ( 2 ) .
blm analogues 35 were prepared by solid - phase synthesis essentially 
 as described using glycosylated threonine building blocks 79 , respectively .
the synthesis of key intermediate 7 was accomplished by condensation of the activated disaccharide ( 18 ) with fmoc - threonine benzyl ester ( 19 ) .
this 
 was converted to 7 in several steps by the introduction 
 of appropriate protecting groups for solid - phase synthesis ( scheme 2 ) .
condensation of 
 gulopyranosyl fluoride 16 with fmoc - threonine benzyl 
 ester ( 19 ) afforded a mixture of anomeric gulopyranosylthreonine 
 derivatives 30 and 37 ( schemes s1 and s2 , supporting information ) , which were separated 
 by flash chromatography on a silica gel column .
intermediate 30 eluted subsequent to 37 from the silica gel 
 column when washed with 2:1 hexanes  ethyl acetate .
following 
 deacetylation to afford 31 , treatment with phenylthioglucoside 29 afforded key intermediate 32 ( scheme s1 , supporting information ) . protecting group optimization 
 then gave intermediate 8 .
the same strategy was used 
 to convert 37 to disaccharide 39 , the latter 
 of which was converted to intermediate 9 ( scheme s2 , supporting information ) .
compounds 79 were then employed for the syntheses of blm 
 analogues 35 by solid - phase synthesis 
 ( scheme 3 ) .
deglycoblm a5 was treated with 1 equiv of cu to form a chelate in 
 which the amino group of the -aminoalanineamide substituent 
 is rendered unreactive , permitting the primary 
 amine at the c - terminus of deglycoblm a5 to react selectively 
 with electrophilic species .
thus , condensation of cu(ii)deglycoblm 
 a5 with the activated p - nitrophenylcarbonate 
 derivative of blm disaccharide ( 27 ) afforded cu(ii)blm 6 , which was demetalated and purified by reversed - phase hplc 
 ( scheme 4 ) .
as shown in figure 3 , fe(ii)blms 3 , 4 , and 5 all effected the relaxation 
 of supercoiled pbr322 
 plasmid dna when used at 4 m concentration , but they did so 
 rather weakly and without producing detectable double - strand cleavage . 

thus , unlike blm itself , which cleaves dna more potently than deglycoblm 
 and with more associated double - strand breaks , introduction of the 
 blm disaccharide by attachment at the threonine moiety actually diminished 
 dna strand scission .
this is consistent with reports that the linker 
 region of blm must assume a specific conformation to exhibit optimal 
 dna cleavage properties ; the introduction of a large substituent 
 to threonine in the form of the disaccharide presumably precludes 
 folding into the conformation optimal for dna cleavage . for fe(ii)blm 6 ,
this experiment employed supercoiled 
 psp64 plasmid dna , also demonstrating that the source of the plasmid 
 dna was unimportant .
furthermore , as 
 might have been anticipated , introduction of the blm saccharide within 
 the c - terminal substituent had no effect on the extent of double - strand 
 cleavage by 6 relative to deglycoblm .
the cytotoxicity 
 of the modified blms was studied in comparison with that of blm a5 and deglycoblm a5 , by use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 
 bromide ( mtt ) as a vital dye to assess cell viability . as shown in 
 figure 5 ,
deglycoblm a5 was significantly 
 less cytotoxic than blm a5 , potentially due to multiple 
 factors including its lower potency as a dna cleaving agent , its propensity 
 to produce fewer double - strand dna breaks , and the absence of the 
 tumor cell targeting disaccharide moiety .
blm analogue 3 was slightly more cytotoxic than deglycoblm a5 at the 
 highest two concentrations tested .
since this analogue cleaves dna 
 no more potently than deglycoblm a5 , its greater cytotoxic 
 potential must be due to the tumor selectivity and uptake associated 
 with the presence of the blm disaccharide in 3 , even 
 though the disaccharide is present in a 
consistent with this interpretation was the very weak cytotoxic 
 activity of blm analogues 4 and 5 , which 
 have disaccharides in the same position as 3 and cleave 
 dna as well as 3 but whose disaccharides lack tumor - targeting 
 properties .
blm analogue 6 , having the blm disaccharide 
 appended 
 to the c - terminal substituent , was intermediate in cytotoxicity between 
 blm a5 and deglycoblm a5 .
since blm 6 cleaved dna with a potency only slightly greater than that of deglycoblm 
 a5 and produced no more double - strand cleavage than deglycoblm 
 a5 , its greater cytotoxicity can most logically be ascribed 
 to the presence of the tumor - selective blm disaccharide .
this was 
 true in spite of the fact that the disaccharide was attached to the 
 molecule in a position quite different than that in blm itself .
this 
 thesis was tested by preparing cyanine dye - labeled conjugates of the 
 cytotoxic blms ( 44 , 45 , 47 , 
 and 55 , corresponding to blms 1 , 2 , 3 , and 6 , respectively ) ( figure 6 ) . as shown in figure 7 , 

the efficiencies of cellular uptake of these four blm analogues were 
 in precisely the same order as their cytotoxic potencies . in 
 the aggregate
, these results strongly support the conclusion 
 that blm is modular , consisting of an aglycon responsible for dna 
 cleavage , connected to a tumor - selective disaccharide .
it may be noted 
 that the foregoing analysis presumes that all of the blm derivatives 
 studied kill du145 cells by the same mechanism involving dna cleavage . 
 while there is no direct evidence in support of this assumption ,
it 
 may be noted that a study of the relative potencies of dna cleavage 
 and cellular cytotoxicity of a number of blm analogues failed to indicate 
 a strict correlation between potency of dna cleavage in vitro and 
 cellular cytotoxicity but provides some general support for this assumption .
the cytotoxicity of blm , which is believed 
 to form the basis for 
 its antitumor activity , is often attributed to its ability to mediate 
 double - strand breaks in dna . while less potent than blm a5 as cytotoxic agents , deglycoblm a5 ( 2 ) and 
 blms 3 and 6 all mediated killing of cultured 
 du145 cells .
the potency of the three compounds as cytotoxins was 
 strongly dependent on the presence of the blm disaccharide moiety 
 somewhere in their structure , and on their potency of supercoiled 
 plasmid dna relaxation .
however , all three compounds exhibited minimal 
 double - strand cleavage of plasmid dna .
an interesting issue not addressed 
 in the present study is whether blms such as 2 , 3 , and 6 possess a mechanism for double - strand 
 cleavage of chromatin , which is not apparent when simpler dnas are 
 used as substrates .
a recent report from our laboratory suggests a 
 mechanism by which this might occur .
specifically , 
 we have shown that dnas strongly bound by feblms undergo double - strand 
 cleavage predominantly by a novel mechanism involving closely spaced 
 independent single - strand cleavages .
it may well be the case that 
 such double - strand cleavage reactions constitute the molecular basis 
 for antitumor activity of blm .
such double - strand cleavage reactions 
 would not be apparent in the analysis provided in figures 3 and 4 .
the broad selectivity 
 for tumor cells noted for blm and its sugars 
 suggests that the targeting must involve some process common to all 
 or most cancers .
one well - known characteristic of tumor cells is the 
 warburg effect , that is , the propensity of tumor cells to produce 
 increased amounts of atp via glycolysis rather than by mitochondrial 
 oxidative phosphorylation .
the shift 
 to glycolysis is accompanied by upregulation of glucose transporters to provide the greater amounts of glucose required 
 to support increased glycolysis .
in fact , treatment of two normal 
 cell lines with the mitochondrial complex i inhibitor rotenone forced 
 these cells to utilize increased glycolysis in the same fashion as 
 tumor cells and resulted in their greatly enhanced ability to incorporate 
 blm - cy5 * * ( 44 ) ( figure 8) . in 
 support of the possible involvement of glucose transporters in the 
 binding and uptake of blm ( sugars ) , it was found ( figure 9 ) that glut1 inhibitors cytochalasin b and phloretin 
 mediated dose - dependent inhibition of the binding / uptake of blm - cy5 * * 
 ( 44 ) by sw-480 colon cancer cells .
at least one sugar 
 transporter ( glut5 ) has been reported to facilitate the cellular uptake 
 of a simple sugar ( fructose ) conjugated to a cyanine dye ( cy5.5 ) .
the effect of adding 
 the blm disaccharide to different positions 
 of deglycoblm was studied .
attachment to the -hydroxyhistidine 
 moiety , as in the natural product , significantly potentiated both 
 dna cleavage and cytotoxicity toward cultured du145 human prostate 
 cancer cells .
when the blm disaccharide was attached to the threonine 
 moiety , dna cleavage activity was significantly diminished but the 
 cytotoxicity toward du145 cells was enhanced nonetheless , arguing 
 that the cytotoxicity of deglycoblm is severely limited by lack of 
 cellular uptake .
two analogues in which close structural analogues 
 of the blm disaccharide were attached to threonine exhibited similar 
 dna cleavage but essentially no cytotoxicity , consistent with the 
 thesis that both facilitated uptake and dna cleavage are essential 
 to the expression of cytotoxicity by bleomycin .
an analogue having 
 the blm saccharide attached to the c - terminal substituent exhibited 
 relatively good dna cleavage and cytotoxicity , entirely consistent 
 with the need for efficient uptake and dna cleavage as prerequisites 
 for cytotoxicity .
the relationship between ( blm sugar - mediated ) cell 
 uptake and cytotoxicity was confirmed by measuring the actual extent 
 of incorporation of cytotoxic blm analogues following conjugation 
 to a cyanine dye .
these findings confirm that blm is a modular molecule , 
 composed of a tumor cell targeting moiety ( the saccharide ) attached 
 to a cytotoxic dna - cleaving domain ( the blm aglycon ) .
this finding 
 implies that the blm saccharide moiety may be able to deliver other 
 cytotoxins selectively to tumor cells .
the uptake mechanism is suggested 
 to involve one or more cell surface receptors involved in glucose 
 transport in tumor cells , which are upregulated in tumor cells to 
 support their enhanced use of glycolysis .
to a stirred solution of 424 mg ( 1.70 mmol ) 
 of methyl trioxorhenium ( mto ) and 4  molecular sieves in 150 
 ml of ch2cl2 were added sequentially 11.9 g 
 ( 126 mmol ) of urea hydrogen peroxide ( uhp ) , 10.0 ml ( 10.6 g , 50.0 
 mmol ) of dibutyl phosphate , and 1.43 g ( 21.0 mmol ) of pyrazole .
the 
 resulting yellow solution was stirred for 10 min , followed by addition 
 of 14.0 g ( 42.0 mmol ) of 3-o - carbamoyl-4,6-di - o - tert - butylsilylene - d - glucal ( 10 ) .
the reaction mixture was 
 stirred for 4 h and then filtered through a pad of celite .
the filtrate 
 was concentrated under diminished pressure , and the residue was purified 
 by flash chromatography on a silica gel column ( 44  4 cm ) .
elution 
 with ethyl acetate gave 11 as a colorless foam : yield 
 7.22 g ( 30% ) ; silica gel thin - layer chromatography ( tlc ) rf 0.58 ( ethyl acetate ) ; [ ]d21 18.8 ( c 0.78 , chcl3 ) ; h nmr ( cdcl3 )  
 0.850.96 ( m , 6h ) , 0.93 ( s , 9h ) , 0.99 ( s , 9h ) , 1.311.38 
 ( m , 4h , j = 7.8 hz ) , 1.561.64 ( m , 4h , j = 7.2 hz ) , 3.433.57 ( m , 2h ) , 3.773.84 
 ( t , 1h , j = 9.6 hz ) , 3.843.91 ( t , 1h , j = 9.9 hz ) , 3.994.12 ( m , 4h ) , 4.144.20 
 ( m , 1h ) , 4.244.29 ( m , 1h ) , 4.864.92 ( t , 1h , j = 9.3 hz ) , 5.0 ( br s , 2h ) , and 5.30 ( t , 1h , j = 7.8 hz ) ; c nmr ( cdcl3 )  13.5 , 18.5 , 
 19.9 , 22.6 , 26.9 , 27.3 , 31.95 , 32.04 , 66.0 , 67.9 , 68.06 , 68.14 , 71.0 , 
 73.2 , 73.3 , 74.1 , 77.8 , 98.8 ( d ) , and 157.8 ; p nmr ( cdcl3 )  1.53 ; mass spectrum ( fast atom bombardment , 
 fab ) m / z 556.1703 ( m + h ) ( c23h47o10nsip requires 556.1706 ) . the reaction was carried out in the same 
 flask as for 11 .
elution of the silica gel column with 
 ethyl acetate gave 12 as a colorless foam : yield 6.38 
 g ( 27% ) ; silica gel tlc rf 0.39 ( ethyl 
 acetate ) ; [ ]d22 + 34.4 ( c 0.84 , chcl3 ) ; h nmr ( cdcl3 )  0.880.93 ( m , 6h ) , 
 0.95 ( s , 9h ) , 1.01 ( s , 9h ) , 1.311.45 ( m , 4h ) , 1.581.69 
 ( m , 4h ) , 3.673.82 ( dt , 1h , j = 9.3 and 8.7 
 hz ) , 3.914.18 ( m , 8h ) , 4.25 ( t , 1h , j = 9.3 
 hz ) , 4.894.96 ( dd , 1h , j = 6.6 and 3.3 hz ) , 
 5.28 ( br s , 2h ) , and 5.585.60 ( dd , 1h , j = 
 5.1 and 1.5 hz ) ; c nmr ( cdcl3 )  13.5 , 
 18.6 , 19.9 , 22.5 , 26.8 , 27.3 , 32.03 , 32.10 , 66.2 , 68.0 , 68.2 , 69.2 , 
 69.5 , 71.3 , 73.0 , 98.0 , and 156.6 ; p nmr ( cdcl3 )  2.03 ; mass spectrum ( fab ) m / z 556.1704 ( m + h ) ( c23h47o10nsip requires 556.1706 ) . to a 
 cooled ( 0 c ) solution containing 0.412 g ( 0.741 mmol ) of 12 in 1.5 ml of pyridine was added 0.140 ml ( 0.151 g , 0.148 
 mmol ) of acetic anhydride .
the reaction mixture was allowed to warm 
 to room temperature and was stirred overnight .
the solution was diluted 
 with 20 ml of etoac and poured into 20 ml of ice water .
the mixture 
 was extracted with three 20 ml portions of etoac , and the combined 
 organic extract was washed sequentially with three 25 ml portions 
 of 0.5 m hcl and 25 ml of saturated aqueous nahco3 .
the 
 organic layer was then dried ( mgso4 ) and concentrated under 
 diminished pressure .
the residue was purified by flash chromatography 
 on a silica gel column ( 36  2 cm ) .
ethyl 
 acetate gave 13 as a colorless oil : yield 0.411 g ( 93% ) ; 
 silica gel tlc rf 0.23 ( 1:1 hexanes  ethyl 
 acetate ) ; [ ]d21 + 11.0 ( c 1.12 , chcl3 ) ; h nmr ( cdcl3 )  0.850.89 ( m , 6h ) , 
 0.92 ( s , 9h ) , 0.99 ( s , 9h ) , 1.281.41 ( m , 4h ) , 1.561.66 
 ( m , 4h ) , 2.08 ( s , 3h ) , 3.814.11 ( m , 8h ) , 5.00 ( br s , 2h ) , 
 5.065.10 ( dd , 1h , j = 6.3 and 3.3 hz ) , 5.25 
 ( dd , 1h , j = 1.8 and 1.8 hz ) , and 5.465.49 
 ( dd , 1h , j = 4.8 and 1.8 hz ) ; c nmr 
 ( cdcl3 )  13.4 , 18.5 , 19.8 , 20.6 , 22.5 , 26.7 , 27.2 , 
 32.0 , 32.1 , 65.9 , 67.8 , 67.9 , 69.0 , 69.8 , 70.6 , 71.6 , 95.2 , 155.7 , 
 and 169.4 ; p nmr ( cdcl3 )  1.91 ; 
 mass spectrum ( fab ) m / z 598.2810 
 ( m + h ) ( c25h49o11nsip 
 requires 598.2813 ) . to a cooled ( 78 
 c ) solution containing 2.62 g ( 4.38 mmol ) of 13 and 4  molecular sieves in 15 ml of ch2cl2 were added sequentially 711 l ( 723 mg , 6.57 mmol ) of thiophenol 
 and 1.98 ml ( 2.43 g , 10.9 mmol ) of tmsotf .
the reaction mixture was 
 allowed to warm to ambient temperature and was quenched by the addition 
 of 10 ml of saturated aqueous nahco3 .
the reaction mixture 
 was diluted with 100 ml of ch2cl2 , washed sequentially 
 with 50 ml of h2o and 50 ml of brine , and then dried ( mgso4 ) and concentrated under diminished pressure .
the residue 
 was purified by flash chromatography on a silica gel column ( 41  
 3 cm ) .
elution with 2:1 hexanes  ethyl acetate gave 14 as colorless needles following crystallization from hot petroleum 
 ether : yield 2.00 g ( 92% ) ; silica gel tlc rf 0.50 ( 2:1 hexanes  ethyl acetate ) ; mp 8991 c ; 
 [ ]d22 + 87.8 ( c 1.08 , chcl3 ) ; h 
 nmr ( cdcl3 )  1.02 ( s , 9h ) , 1.05 ( s , 9h ) , 2.12 ( s , 
 3h ) , 3.96 ( t , 1h , j = 10.2 hz ) , 4.06 ( m , 1h ) , 4.19 
 ( t , 1h , j = 9.6 hz ) , 4.37 ( m , 1h ) , 5.10 ( dd , 1h , j = 6.3 and 3.3 hz ) , 5.11 ( br s , 2h ) , 5.40 ( s , 1h ) , 5.50 
 ( d , 1h , j = 3.3 hz ) , 7.267.33 ( m , 3h ) , and 
 7.44 ( m , 2h ) ; c nmr ( cdcl3 )  20.0 , 
 20.9 , 22.6 , 26.9 , 27.3 , 66.1 , 68.7 , 71.8 , 72.2 , 72.4 , 86.4 , 127.8 , 
 129.1 , 131.7 , 133.2 , 155.9 , and 169.8 ; mass spectrum ( fab ) m / z 498.1984 ( m + h ) ( c23h36o7nsis requires 498.1982 ) .
to a cooled 
 ( 0 c ) solution containing 1.00 g ( 1.97 mmol ) of 3,4,6-tri - o - benzyl-1,2-o-(1-methoxyethylidene)--l - gulopyranose ( 15 ) in 20 ml of dry ch2cl2 was added dropwise 
 287 l ( 350 mg , 2.17 mmol ) of dast .
the solution was allowed 
 to warm slowly to ambient temperature and stirred for 3 h. the solution 
 was then diluted with 50 ml of ch2cl2 and poured 
 into 50 ml of ice water .
the aqueous layer was extracted with three 
 25 ml portions of ch2cl2 , and the combined organic 
 extract was washed sequentially with 50 ml of h2o , 50 ml 
 of saturated aqueous nahco3 , and 50 ml of brine and then 
 dried ( mgso4 ) and concentrated under diminished pressure . 

the residue was purified by flash chromatography on a silica gel column 
 ( 36  2 cm ) .
elution with 7:3 hexanes  ethyl acetate gave 16 as a pale yellow oil : yield 927 mg ( 95% ) ; silica gel tlc rf 0.84 ( 1:1 hexanes  ethyl acetate ) ; [ ]d21 + 41.4 ( c 0.97 , chcl3 ) ; h nmr ( cdcl3 )  2.09 ( s , 3h ) , 3.633.74 ( m , 1h ) , 4.07 ( q , 1h , j = 3.9 hz ) , 4.34 ( dt , 1h , j = 4.5 and 
 1.8 hz ) , 4.824.63 ( m , 6h ) , 5.16 ( m , 1h ) , 5.54 ( d , 0.5h , j = 7.2 hz ) , 5.72 ( d , 1h , j = 7.2 hz ) , 
 and 7.267.39 ( m , 15h ) ; c nmr ( cdcl3 )  20.8 , 68.2 , 70.3 ( 0.5c ) , 70.6 ( 0.5c ) , 72.6 , 73.2 , 73.4 , 
 73.7 ( 0.5c ) , 73.8 ( 0.5c ) , 104.5 ( 0.5c ) , 107.3 ( 0.5c ) , 127.7 , 127.8 , 
 127.9 , 128.1 , 128.3 , 128.4 , 137.28 , 137.32 , 137.7 , and 169.7 ; mass 
 spectrum ( fab ) m / z 493.2029 ( m  
 h ) ( c29h30o6f requires 
 493.2026 ) . to a solution 
 of 394 mg ( 797 mol ) of acetylated glycosyl fluoride 16 in 10 ml of dry meoh was added 16.0 mg ( 119 mol ) of k2co3 .
the residue was purified by flash chromatography 
 on a silica gel column ( 34  1 cm ) .
ethyl 
 acetate gave 17 as a pale yellow oil : yield 348 mg ( 97% ) ; 
 silica gel tlc rf 0.71 ( 1:1 hexanes  ethyl 
 acetate ) ; [ ]d21 3.26 ( c 0.38 , chcl3 ) ; h nmr ( cdcl3 )  2.57 ( d , 1h , j = 8.1 hz ) , 3.69 ( m , 3h ) , 3.87 ( m , 2h ) , 4.22 ( dt , 1h , j = 4.8 and 1.8 hz ) , 4.484.62 ( m , 6h ) , 5.29 ( d , 0.5h , j = 6.6 hz ) , 5.47 ( d , 0.5h , j = 6.6 hz ) , 
 and 7.257.41 ( m , 15h ) ; c nmr ( cdcl3 )  68.1 , 68.6 ( 0.5c ) , 68.9 ( 0.5c ) , 72.67 , 72.76 ( 0.5c ) , 72.80 
 ( 0.5c ) , 73.0 , 73.2 , 73.4 , 76.3 ( 0.5c ) , 76.4 ( 0.5c ) , 107.2 ( 0.5c ) , 
 110.0 ( 0.5c ) , 127.66 , 127.76 , 127.81 , 127.98 , 128.05 , 128.11 , 128.3 , 
 128.4 , 128.5 , 137.2 , 137.3 , and 137.7 ; mass spectrum ( fab ) , m / z 451.1920 ( m  h ) ( c27h28o5f requires 451.1921 ) . to a cooled 
 ( 78 c ) solution containing 4  molecular sieves , 
 625 mg ( 1.38 mmol ) of gulosyl fluoride 17 , and 687 mg 
 ( 1.38 mmol ) of phenylthiomannoside 14 in 8 ml of ch2cl2 were added 466 mg ( 2.07 mmol ) of n - iodosuccinimide and 71.0 mg ( 276 mol ) of agotf in the dark . 

the reaction mixture was allowed to warm to 5 c and 
 was quenched with 10 ml of saturated aqueous nahco3 90 
 s after the color changed from colorless to deep purple .
the reaction 
 mixture was diluted with 25 ml of ch2cl2 and 
 poured into 25 ml of saturated aqueous na2s2o3 .
the combined organic extract was 
 washed with 50 ml of h2o and then dried ( mgso4 ) and concentrated under diminished pressure .
the residue was purified 
 by flash chromatography on a silica gel column ( 36  2 cm ) .
elution 
 with 2:1 hexanes  ethyl acetate gave 18 as a colorless 
 foam : yield 1.05 g ( 91% ) ; silica gel tlc rf 0.51 ( 2:1 hexanes  ethyl acetate ) ; [ ]d22 + 8.9 ( c 0.39 , 
 chcl3 ) ; h nmr ( cdcl3 )  0.96 
 ( s , 9h ) , 1.05 ( s , 9h ) , 2.17 ( s , 3h ) , 3.53 ( br s , 1h ) , 3.62 ( m , 2h ) , 
 3.79 ( m , 1h ) , 3.88 ( m , 2h ) , 4.11 ( m , 4h ) , 4.25 ( t , 1h , j = 6.6 hz ) , 4.394.70 ( m , 6h ) , 4.80 ( s , 2h ) , 5.21 ( m , 2h ) , 
 5.45 ( d , 0.5h , j = 6.9 hz ) , 5.63 ( d , 0.5h , j = 6.9 hz ) , 7.14 ( m , 2h ) , and 7.267.36 ( m , 13h ) ; c nmr ( cdcl3 )  19.9 , 20.9 , 22.5 , 26.8 , 
 27.2 , 66.1 , 67.4 , 68.1 , 70.7 , 71.4 , 72.1 , 72.8 , 73.0 , 73.1 , 73.4 , 
 73.6 , 74.2 , 95.2 , 105.1 ( 0.5c ) , 108.0 ( 0.5c ) , 127.6 , 127.7 , 127.8 , 
 127.9 , 128.0 , 128.27 , 128.36 , 128.41 , 137.5 , 137.7 , 156.0 , and 170.0 ; 
 mass spectrum ( fab ) m / z 862.3627 
 ( m + na ) ( c44h58o12nsifna 
 requires 862.3610 ) . to a cooled 
 ( 15 c ) solution containing 4  molecular sieves , 
 187 mg ( 433 mol ) of fmoc - l - threonine benzyl ester 
 ( 19 ) , and 400 mg ( 476 mol ) of fluorinated disaccharide 15 in 3 ml of ch2cl2
were added 380 
 mg ( 1.30 mmol ) of cp2zrcl2 and 334 mg ( 1.30 
 mmol ) of agotf .
the reaction mixture was stirred at 15 c 
 for 3 h until complete consumption of the starting glycosyl fluoride 
 was observed .
the solution was filtered through a pad of celite , and 
 the filtrate was concentrated under a stream of n2 .
the 
 residue was purified by flash chromatography on a silica gel column 
 ( 48  3 cm ) .
elution with 2:1 hexanes  ethyl acetate gave 20 as a colorless foam : yield 376 mg ( 68% ) ; silica gel tlc rf 0.74 ( 1:1 hexanes  ethyl acetate ) ; [ ]d21 15.7 
 ( c 0.17 , chcl3 ) ; h nmr ( cdcl3 )  0.98 ( s , 9h ) , 1.07 ( s , 9h ) , 1.35 ( d , 3h , j = 6.0 hz ) , 2.16 ( s , 3h ) , 3.523.55 ( m , 2h ) , 3.65 
 ( d , 1h , j = 2.7 hz ) , 3.82 ( s , 1h ) , 3.88 ( dd , 1h , j = 5.4 and 4.2 hz ) , 3.97 ( m , 1h ) , 4.05 ( t , 1h , j = 3.6 hz ) , 4.114.25 ( m , 6h ) , 4.334.56 
 ( m , 10h ) , 4.66 ( s , 1h ) , 4.78 ( s , 1h ) , 4.96 ( d , 1h , j = 3.6 hz ) , 5.14 ( d , 1h , j = 2.1 hz ) , 5.195.25 
 ( m , 2h ) , 6.53 ( d , 1h , j = 8.1 hz ) , 7.207.34 
 ( m , 24h ) , 7.61 ( d , 2h , j = 7.5 hz ) , and 7.72 ( d , 
 2h , j = 7.5 hz ) ; c nmr ( cdcl3 )  16.6 , 19.8 , 21.0 , 22.6 , 26.8 , 27.3 , 47.0 , 59.2 , 65.7 , 66.3 , 
 67.0 , 67.2 , 68.0 , 68.5 , 70.3 , 70.7 , 70.8 , 71.4 , 72.1 , 72.5 , 72.8 , 
 73.0 , 73.2 , 74.4 , 77.2 , 95.1 , 95.6 , 119.7 , 125.3 , 125.4 , 127.0 , 127.4 , 
 127.5 , 127.7 , 127.9 , 128.0 , 128.1 , 128.2 , 128.4 , 128.65 , 128.71 , 137.7 , 
 137.95 , 138.02 , 141.1 , 144.0 , 144.1 , 155.6 , 157.0 , 169.8 , and 170.5 ; 
 mass spectrum ( fab ) m / z 1251.5475 
 ( m + h ) ( c70h83o17n2si requires 1251.5461 ) . to a solution of 180 mg ( 143 
 mol ) of threonine 20 in 3 ml of acetonitrile in 
 a plastic tube was carefully added 100 l ( 5.00 mmol ) of 48% 
 hf in h2o .
the solution was stirred overnight and then 
 poured slowly into a mixture of 20 g of ice and 20 ml of saturated 
 aqueous nahco3 .
the aqueous layer was extracted with three 
 50 ml portions of ethyl acetate , and the combined organic extract 
 was washed with 50 ml of h2o .
the organic layer was then 
 dried ( mgso4 ) and concentrated under diminished pressure . 

the residue was purified by flash chromatography on a silica gel column 
 ( 38  1 cm ) .
acetic 
 acid gave 21 as a colorless foam : yield 0.142 g ( 89% ) ; 
 silica gel tlc rf 0.50 ( 88:10:2 chloroform  methanol 
acetic 
 acid ) ; [ ]d22 5.2 ( c 0.14 , chcl3 ) ; h nmr ( cdcl3 )  1.18 ( d , 2h , j = 
 6.0 hz ) , 1.28 ( br s , 1h ) , 2.08 ( s , 3h ) , 3.51 ( dd , 1h , j = 5.4 and 4.5 hz ) , 3.64 ( m , 1h ) , 3.763.94 ( m , 7h ) , 4.09 
 ( br s , 2h ) , 4.17 ( m , 1h ) , 4.23 ( t , 1h , j = 6.3 hz ) , 
 4.404.59 ( m , 9h ) , 4.64 ( br s , 2h ) , 4.99 ( br s , 3h ) , 5.10 ( d , 
 1h , j = 8.4 hz ) , 5.125.24 ( q , 2h , j = 12.3 and 11.7 hz ) , 5.35 ( s , 1h ) , 6.72 ( d , 1h , j = 8.1 hz ) , 7.197.40 ( m , 24h ) , 7.65 ( d , 2h , j = 6.9 hz ) , and 7.75 ( d , 2h , j = 7.5 hz ) ; c nmr ( cdcl3 )  15.3 , 20.9 , 29.6 , 47.1 , 
 59.0 , 62.6 , 66.7 , 67.5 , 68.7 , 70.1 , 71.1 , 71.8 , 72.4 , 72.7 , 73.2 , 
 73.4 , 93.1 , 97.2 , 119.8 , 125.0 , 125.2 , 127.1 , 127.5 , 127.6 , 127.7 , 
 127.8 , 128.0 , 128.1 , 128.3 , 135.3 , 137.8 , 138.0 , 141.1 , 143.8 , 143.9 , 
 156.4 , 157.1 , 169.8 , and 171.0 ; mass spectrum ( fab ) m / z 1111.4447 ( m + h ) ( c62h67o17n2 requires 1111.4439 ) . to a cooled ( 0 c ) solution 
 containing 0.102 g ( 0.092 mmol ) of 21 in 2 ml of pyridine 
 was added 0.043 ml ( 0.047 g , 0.485 mmol ) of acetic anhydride .
the 
 reaction mixture was allowed to warm to ambient temperature and then 
 stirred overnight .
the solution was diluted with 25 ml of etoac and 
 poured into 10 ml of ice water .
the aqueous layer was extracted with 
 three 25 ml portions of etoac , and the combined organic extract was 
 washed sequentially with three 50 ml portions of 0.5 m hcl , 50 ml 
 of h2o , and 50 ml of brine and then dried ( mgso4 ) and concentrated under diminished pressure .
the residue was purified 
 by flash chromatography on a silica gel column ( 48  2 cm ) .
elution 
 with 1:1 hexanes  ethyl acetate gave 22 as a colorless 
 oil : yield 0.109 g ( 99% ) ; silica gel tlc rf 0.55 ( 1:1 hexanes  ethyl acetate ) ; [ ]d21 17.4 ( c 0.15 , chcl3 ) ; h nmr ( cdcl3 )  
 1.33 ( d , 3h , j = 6.0 hz ) , 2.01 ( s , 3h ) , 2.06 ( s , 
 3h ) , 2.15 ( s , 3h ) , 3.483.59 ( m , 2h ) , 3.69 ( d , 1h , j = 2.4 hz ) , 3.85 ( t , 1h , j = 3.3 hz ) , 
 4.05 ( m , 3h ) , 4.214.52 ( m , 10h ) , 4.564.68 ( m , 3h ) , 
 4.83 ( d , 3h , j = 3.6 hz ) , 4.99 ( d , 1h , j = 3.6 hz ) , 5.085.19 ( m , 2h ) , 5.30 ( m , 3h ) , 6.43 ( d , 1h , j = 8.1 hz ) , 7.197.37 ( m , 24h ) , 7.60 ( d , 2h , j = 7.5 hz ) , and 7.72 ( d , 2h , j = 7.5 hz ) ; c nmr ( cdcl3 )  16.6 , 20.59 , 20.64 , 20.9 , 
 47.0 , 59.2 , 62.3 , 65.7 , 67.0 , 67.2 , 68.1 , 69.0 , 69.8 , 69.9 , 72.0 , 
 72.2 , 72.7 , 73.0 , 73.2 , 74.4 , 94.8 , 96.1 , 119.7 , 125.2 , 125.3 , 126.95 , 
 126.99 , 127.48 , 127.55 , 127.64 , 127.8 , 127.9 , 128.00 , 128.1 , 128.2 , 
 128.4 , 135.4 , 137.6 , 137.9 , 141.1 , 143.8 , 144.0 , 155.1 , 157.0 , 169.7 , 
 169.9 , 170.4 , and 170.5 ; mass spectrum ( fab ) m / z 1195.4641 ( m + h ) ( c66h71o19n2 requires 1195.4651 ) . to a solution 
 containing 0.101 g ( 0.085 mmol ) of threonine disaccharide 22 in 2 ml of n , n - dimethylformamide 
 ( dmf ) were added sequentially 0.034 g ( 0.592 mmol ) of kf , 0.025 ml 
 ( 0.018 g , 0.179 mmol ) of et3n , and 0.024 g ( 0.111 mmol ) 
 of ( boc)2o .
the reaction mixture was stirred at ambient 
 temperature until complete consumption of the starting material was 
 observed by silica gel tlc analysis .
the solution was diluted with 
 25 ml of etoac and washed with 10 ml of h2o .
the aqueous 
 layer was back - extracted with three 10 ml portions of etoac , and the 
 combined organic extract was washed sequentially with 25 ml of saturated 
 aqueous nahco3 and 25 ml of brine and then dried ( mgso4 ) and concentrated under diminished pressure .
the residue 
 was purified by flash chromatography on a silica gel column ( 46  
 2 cm ) .
elution with 2:1 hexanes  ethyl acetate gave 23 as a colorless oil : yield 0.078 g ( 86% ) ; silica gel tlc rf 0.68 ( 3:1 etoac  hexanes ) ; [ ]d22 18.1 
 ( c 0.16 , chcl3 ) ; h nmr ( cdcl3 )  1.25 ( d , 3h , j = 5.7 hz ) , 1.40 
 ( s , 9h ) , 2.05 ( s , 3h ) , 2.07 ( s , 3h ) , 2.15 ( s , 3h ) , 3.443.55 
 ( m , 2h ) , 3.66 ( s , 1h ) , 3.82 ( t , 1h , j = 3.3 hz ) , 
 3.98 ( t , 1h , j = 3.3 hz ) , 4.014.13 ( m , 2h ) , 
 4.21 ( t , 1h , j = 6.0 hz ) , 4.334.49 ( m , 7h ) , 
 4.564.68 ( m , 2h ) , 4.83 ( s , 2h ) , 4.95 ( d , 1h , j = 3.6 hz ) , 5.08 ( s , 2h ) , 5.29 ( m , 3h ) , 5.76 ( d , 2h , j = 8.4 hz ) , 7.17 ( m , 2h ) , and 7.267.30 ( m , 18h ) ; c nmr ( cdcl3 )  16.3 , 20.6 , 20.8 , 20.9 , 28.2 , 58.7 , 
 62.3 , 65.8 , 66.9 , 68.2 , 68.9 , 69.9 , 72.2 , 72.7 , 72.8 , 73.0 , 73.2 , 
 74.8 , 79.7 , 94.6 , 96.6 , 127.5 , 127.6 , 127.9 , 128.1 , 128.2 , 128.4 , 
 135.5 , 137.7 , 138.0 , 138.2 , 155.1 , 156.3 , 169.8 , 169.9 , 170.5 , and 
 170.6 ; mass spectrum ( fab ) m / z 1073.4507 
 ( m + h ) ( c56h69o19n2 requires 1073.4495 ) .
a suspension containing 150 
 mg ( 140 mol ) of threonine 
 disaccharide 23 and 20 mg of pd(oh)2/c in 
 10 ml of 1:1 tetrahydrofuran ( thf)meoh was purged with h2 and maintained under a h2 atmosphere overnight . 
 the reaction mixture was then diluted with meoh and filtered through 
 a pad of celite , and the filtrate was concentrated under diminished 
 pressure .
the residue was purified by flash chromatography on a silica 
 gel column ( 39  1 cm ) .
acetic 
 acid gave 24 as a colorless solid : yield 94 mg ( 95% ) ; 
 silica gel tlc rf 0.45 ( 3:2 ethyl acetate  methanol ) ; 
 [ ]d22 + 1.7 ( c 0.24 , meoh ) ; h nmr ( methanol - d4 )  1.12 ( d , 3h , j = 
 4.8 hz ) , 1.35 ( s , 9h ) , 1.94 ( s , 3h ) , 1.95 ( s , 3h ) , 2.03 ( s , 3h ) , 3.65 
 ( m , 3h ) , 3.904.01 ( m , 6h ) , 4.084.19 ( m , 2h ) , 4.34 
 ( br s , 1h ) , 4.90 ( d , 1h , j = 3.3 hz ) , 4.95 ( s , 1h ) , 
 5.15 ( m , 1h ) , and 5.27 ( s , 1h ) ; c nmr ( methanol - d4 )  16.1 , 20.7 , 20.8 , 22.1 , 28.8 , 61.2 , 
 62.9 , 63.7 , 67.6 , 68.0 , 69.7 , 70.5 , 70.8 , 71.2 , 71.5 , 74.6 , 80.4 , 
 96.1 , 97.0 , 158.2 , 158.4 , 171.4 , 171.6 , 172.4 , and 177.7 ; mass spectrum 
 ( fab ) m / z 735.2430 ( m + na ) ( c28h44o19n2na 
 requires 735.2436 ) . to a solution containing 
 140 mg ( 196 mol ) of 24 in 2 ml of ch2cl2 were added 0.2 ml of me2s and 2 ml of cf3cooh .
the reaction mixture was maintained at ambient temperature 
 until complete consumption of the starting material was observed by 
 silica gel tlc analysis .
the reaction mixture was diluted with 5 ml 
 of toluene and then concentrated under diminished pressure .
the residue 
 was then dissolved in 2 ml of saturated aqueous nahco3 . 
 to the solution
was added a solution containing 67.0 mg ( 200 mol ) 
 of fmocosu in 2 ml of ch3cn .
the reaction mixture was stirred 
 under n2 for 8 h and then diluted with 25 ml of etoac . 

the solution was washed with 25 ml of 0.5 m hcl , and the aqueous layer 
 was back - extracted with three 25 ml portions of etoac .
the combined 
 organic extract was then dried ( mgso4 ) and concentrated 
 under diminished pressure .
purification by flash chromatography on 
 a silica gel column gave 7 as a colorless solid : yield 
 114 mg ( 70% over two steps ) ; silica gel tlc rf 0.17 ( 85:13:2 chloroform  methanol  acetic acid ) ; [ ]d21 + 23.4 ( c 0.10 , meoh ) ; h nmr ( methanol - d4 )  1.31 ( d , 3h , j = 6.3 hz ) , 
 2.05 ( s , 3h ) , 2.13 ( s , 3h ) , 2.30 ( s , 3h ) , 3.75 ( d , 2h , j = 5.7 hz ) , 3.87 ( d , 1h , j = 3.0 hz ) , 4.074.16 
 ( m , 5h ) , 4.224.39 ( m , 5h ) , 4.50 ( dd , 1h , j = 3.6 and 2.4 hz ) , 5.02 ( d , 1h , j = 3.3 hz ) , 5.09 
 ( d , 1h , j = 1.2 hz ) , 5.29 ( d , 2h ) , 5.42 ( m , 1h ) , 
 7.107.20 ( m , 2h ) , 7.30 ( t , 2h , j = 7.2 hz ) , 
 7.37 ( t , 2h , j = 6.9 hz ) , 7.70 ( d , 2h , j = 7.2 hz ) , and 7.76 ( d , 2h , j = 6.9 hz ) ; c nmr ( methanol - d4 )  16.6 , 20.7 , 
 60.2 , 62.5 , 63.7 , 67.5 , 67.6 , 68.3 , 69.0 , 70.4 , 70.8 , 71.20 , 71.23 , 
 71.4 , 74.7 , 96.7 , 97.1 , 120.9 , 126.3 , 126.4 , 128.2 , 128.8 , 129.2 , 
 129.9 , 142.5 , 145.2 , 145.3 , 158.1 , 159.3 , 171.4 , 171.6 , 172.5 , and 
 174.1 ; mass spectrum ( electrospray ) m / z 835.2766 ( m + h ) ( c38h47o19n2 requires 835.2773 ) . to 0.366 g ( loading 
 of 0.082 mmol / g ) of swollen novasyn tentagel amino resin functionalized 
 with the fmoc - protected bithiazole moiety and blm a6 c - substituent 
 ( scheme 3 ) was added 
 a 20% solution of piperidine in 2 ml of dmf .
the reaction mixture 
 was shaken for 5 min , and the solvent was removed by filtration .
the 
 resin was washed successively with three 10 ml portions of ch2cl2 , three 10 ml portions of meoh , and three 10 
 ml portions of dmf .
the piperidine deblocking step was repeated twice 
 and the resin was washed successively with three 10 ml portions of 
 ch2cl2 , three 10 ml portions of meoh , and three 
 10 ml portions of dmf .
an aliquot of the resin was removed , and a 
 qualitative kaiser free amine test indicated the presence of a free 
 amine .
the resin was suspended in a solution of 0.068 g ( 0.180 mmol ) 
 of 2-(1h - benzotriazol-1-yl)-1,1,3,3-tetramethyluronium 
 hexafluorophosphate ( hbtu ) , 0.150 g ( 0.180 mmol ) of threonine disaccharide 7 , 0.027 g ( 0.180 mmol ) of hydroxybenzotriazole ( hobt ) , and 
 0.063 ml ( 0.360 mmol ) of n , n - diisopropylethylamine 
 ( dipea ) in 2 ml of dmf .
the reaction mixture was shaken for 45 min , 
 and the solvent was removed by filtration .
the resin was washed successively 
 with three 10 ml portions of ch2cl2 , three 10 
 ml portions of meoh , and three 10 ml portions of dmf .
an aliquot of 
 the resin was removed , and a qualitative kaiser free amine test indicated 
 the absence of a free amine .
an aliquot of the resin was subjected 
 to fmoc cleavage assay analysis , which indicated a loading of 0.032 
 mmol / g
the resin was dried 
 under diminished pressure and carried on to the methylvalerate coupling 
 step : mass spectrum ( matrix - assisted laser desorption ionization time - of - flight , 
 maldi - tof ) m / z 1108.3 ( m + h ) ( theoretical m / z 1108.3 ) . to 0.355 g ( loading 
 of 0.032 mmol / g ) of swollen tripeptide resin
the reaction mixture was shaken for 
 5 min , and the solvent was removed by filtration .
the resin was washed 
 successively with three 10 ml portions of ch2cl2 , three 10 ml portions of meoh , and three 10 ml portions of dmf . 

the piperidine deblocking step was repeated twice and the resin was 
 washed successively with three 10 ml portions of ch2cl2 , three 10 ml portions of meoh , and three 10 ml portions of 
 dmf .
an aliquot of the resin was removed , and a qualitative kaiser 
 free amine test indicated the presence of a free amine .
the resin 
 was suspended in a solution of 0.021 g ( 0.057 mmol ) of fmoc - methylvalerate , 0.022 g ( 0.057 mmol ) of hbtu , 0.009 g ( 0.057 mmol ) of hobt , and 
 0.020 ml ( 0.114 mmol ) of hunig s base ( dipea ) in 2 ml of dmf . 

the reaction mixture was stirred for 45 min , and the solvent was removed 
 by filtration .
the resin was washed successively with three 10 ml 
 portions of ch2cl2 , three 10 ml portions of 
 meoh , and three 10 ml portions of dmf .
an aliquot of the resin was 
 removed , and a qualitative kaiser free amine test indicated the absence 
 of a free amine .
an aliquot of the resin was subjected to fmoc cleavage 
 assay analysis , which indicated a loading of 0.029 mmol / g ( corresponding 
 to 94% coupling efficiency ) .
the resin was dried under diminished 
 pressure and carried on to the histidine coupling step : mass spectrum 
 ( maldi - tof ) m / z 1237.4 ( m + h ) ( theoretical m / z 1237.4 ) . to 0.350 g ( loading 
 of 0.029 mmol / g ) of swollen tetrapeptide resin
the reaction mixture was shaken for 
 10 min , and then the solvent was removed by filtration .
the resin 
 was washed successively with three 10 ml portions of ch2cl2 , three 10 ml portions of meoh , and three 10 ml portions 
 of dmf .
the piperidine deblocking step was repeated twice and the 
 resin was washed successively with three 10 ml portions of ch2cl2 , three 10 ml portions of meoh , and three 10 
 ml portions of dmf .
an aliquot of the resin was removed , and a qualitative 
 kaiser free amine test indicated the presence of a free amine .
the 
 resin was suspended in a solution of 0.031 g ( 0.050 mmol ) of fmoc - tritylhistidine , 
 0.019 g ( 0.050 mmol ) of 2-(7-aza-1h - benzotriazol-1-yl)-1,1,3,3-tetramethyluronium 
 hexafluorophosphate ( hatu ) , 0.007 g ( 0.050 mmol ) of 1-hydroxy-7-azabenzotriazole 
 ( hoat ) , and 0.017 ml ( 0.100 mmol ) of hunig s base in 2 ml of 
 dmf .
the reaction mixture was stirred for 45 min , and the solvent 
 was removed by filtration .
the resin was washed successively with 
 three 10 ml portions of ch2cl2 , three 10 ml 
 portions of meoh , and three 10 ml portions of dmf .
an aliquot of the 
 resin was removed , and a qualitative kaiser free amine test indicated 
 the absence of a free amine .
an aliquot of the resin was subjected 
 to fmoc cleavage assay analysis , which indicated a loading of 0.051 
 mmol / g .
the resin was dried under diminished pressure : mass spectrum 
 ( maldi - tof ) m / z 1616.5 ( m + h ) ( theoretical m / z 1616.5 ) . to a suspension 
 containing 0.118 g of pentapeptide resin
were added three 0.5 ml solutions 
 containing 20% piperidine in dmf for 10 min each .
the resulting resin 
 was washed for 30 s each with seven 5 ml portions of dmf , five 5 ml 
 portions of ch2cl2 , and three 5 ml portions 
 of dmf .
the resin was then added to a 10 ml round - bottom flask containing 
 1 ml of dmf and cooled to 0 c for 10 min . a mixture containing 
 5.00 mg ( 11.6 mol ) of boc - pyrimidoblamic acid and 19.0 mg ( 34.8 mol ) of ( benzotriazol-1-yloxy)tripyrrolidinophosphonium 
 hexafluorophosphate ( pybop )
the reaction mixture was cooled for an additional 10 
 min , followed by the addition of 12 l ( 9.0 mg , 70 mol ) 
 of hunig s base .
after 16 h , the resin was filtered and washed 
 for 30 s each with three 5 ml portions of dmf , three 5 ml portions 
 of ch2cl2 , and three 5 ml portions of meoh . 

the resin was dried under vacuum for 2 h. to a suspension containing 
 0.076 g of resin - bound fully protected deglycoblm a6 precursor 
 was added a solution containing 200 l of triisopropylsilane 
 and 200 l of methyl sulfide . after 5 min , 4 ml of trifluoroacetic 
 acid ( tfa ) was added to the suspension . after 4 h
, the resin was filtered 
 and washed with five 3 ml portions of ch2cl2 and three 3 ml portions of dmf .
the resulting resin was treated 
 with 0.5 ml of a solution containing 20% hydrazine in dmf .
the resin 
 was filtered and then treated with three 0.5 ml portions of 20% hydrazine 
 in dmf for an additional 10 min each .
the resulting oil was dissolved 
 in 0.1% aqueous cf3cooh , frozen , and lyophilized .
the crude 
 product was purified on an alltech alltima c18 reversed - phase 
 semipreparative ( 250  10 mm , 5 m ) hplc column by use 
 of 0.1% aqueous cf3cooh and ch3cn mobile phases . 

 ch3cn ) over a period of 30 min at a flow rate of 4 ml / min .
fractions 
 containing the desired product eluted at 13.2 min and were collected , 
 frozen , and lyophilized to give 3 as a colorless foam : 
 yield 1.1 mg ( 12% ) ; h nmr ( cdcl3 )  0.93 
 ( d , 3h , j = 7.0 hz ) , 1.05 ( d , 3h , j = 6.5 hz ) , 1.06 ( d , 3h , j = 7.5 hz ) , 1.66 ( m , 4h ) , 
 1.901.97 ( m , 7h ) , 2.542.60 ( m , 3h ) , 2.933.04 
 ( m , 10h ) , 3.15 ( dd , 1h , j = 7.5 and 7.0 hz ) , 3.20 
 ( t , 1h , j = 4.5 hz ) , 3.41 ( m , 1h ) , 3.543.59 
 ( m , 4h ) , 3.583.66 ( m , 2h ) , 3.733.79 ( m , 4h ) , 3.88 
 ( t , 1h , j = 6.0 hz ) , 3.94 ( t , 1h , j = 4.0 hz ) , 4.02 ( m , 2h ) , 4.15 ( m , 1h ) , 4.39 ( d , 1h , j = 3.5 hz ) , 4.49 ( d , 1h , j = 2.5 hz ) , 4.57 ( d , 1h , j = 1.5 hz ) , 4.96 ( d , 1h , j = 4.0 hz ) , 
 7.24 ( s , 1h ) , 7.94 ( s , 1h ) , 8.10 ( s , 1h ) , and 8.55 ( s , 1h ) ; mass spectrum 
 ( electrospray ) m / z 1481.5 ( m + h ) , theoretical m / z 1481.7 ; 
 mass spectrum ( tof es+ ) m / z 1481.6633 
 ( m+h ) ( c60h97n20o20s2 requires 1481.6631 ) .
the crude 
 product from the solid - phase synthesis ( supporting 
 information ) was purified on an alltech alltima c18 reversed - phase semipreparative ( 250  10 mm , 5 m ) hplc 
 column by the use of 0.1% cf3cooh and ch3cn 
 mobile phases .
ch3cn ) over a period of 30 min 
 at a flow rate of 4 ml / min .
fractions containing the desired product 
 eluted at 13.3 min and were collected , frozen , and lyophilized to 
 give 4 as a colorless foam : yield 2.0 mg ( 22% ) ; h nmr ( cdcl3 )  1.00 ( d , 3h , j = 6.3 hz ) , 1.13 ( d , 3h , j = 4.8 hz ) , 1.15 ( d , 3h , j = 5.7 hz ) , 1.75 ( m , 5h ) , 1.982.07 ( m , 8h ) , 2.62 
 ( m , 2h ) , 2.98 ( m , 2h ) , 3.033.14 ( m , 12h ) , 3.203.30 
 ( m , 6h ) , 3.40 ( t , 2h , j = 8.7 hz ) , 3.50 ( m , 6h ) , 
 3.643.68 ( m , 5h ) , 3.693.73 ( m , 3h ) , 3.813.96 
 ( m , 8h ) , 5.06 ( m , 2h ) , 4.13 ( t , 1h , j = 3.0 hz ) , 
 4.27 ( m , 2h ) , 4.46 ( d , 1h , j = 2.4 hz ) , 4.65 ( m , 
 4h ) , 5.17 ( d , 1h , j = 3.3 hz ) , 7.32 ( s , 1h ) , 8.02 
 ( d , 1h , j = 0.9 hz ) , 8.18 ( d , 1h , j = 1.2 hz ) , and 8.63 ( s , 1h ) ; mass spectrum ( electrospray ) m / z 1481.6 ( m + h ) , theoretical m / z 1481.7 ; mass spectrum ( tof es+ ) , m / z 1481.6631 ( m + h ) ( c60h97n20o20s2 requires 1481.6631 ) .
the crude 
 product from the solid - phase synthesis ( supporting 
 information ) was purified on an alltech alltima c18 reversed - phase semipreparative ( 250  10 mm , 5 m ) hplc 
 column by use of 0.1% cf3cooh and ch3cn mobile 
 phases .
ch3cn ) over a period of 30 min at a 
 flow rate of 4 ml / min .
fractions containing the desired product eluted 
 at 14.0 min and were collected , frozen , and lyophilized to give 5 as a colorless foam : yield 0.2 mg ( 2% ) ; mass spectrum ( electrospray ) m / z 1481.6 ( m + h ) , theoretical m / z 1481.7 .
to a solution of 83.0 
 mg ( 0.12 mmol ) of 1,3,4,6-tetra - o - acetyl-2-o-(2,4,6-tri - o - acetyl-3-o - carbamoyl--d - mannopyranosyl)--l - gulopyranoside 
 ( 25 ) in 2 ml of dry dmf was added 15.0 mg ( 0.16 mmol ) 
 of the acetate salt of hydrazine at 0 c .
the reaction mixture 
 was warmed to room temperature and stirred for 1 h , at which time 
 analysis by silica gel tlc indicated that the starting material had 
 been completely consumed .
the reaction mixture was diluted with 50 
 ml of etoac , washed with three 15 ml portions of brine , dried over 
 na2so4 , filtered , and concentrated under diminished 
 pressure .
the residue was purified by flash chromatography on a silica 
 gel column ( 20  2 cm ) .
ethyl 
 acetate afforded 26 as a colorless oil : yield 55 mg ( 70% ) ; 
 silica gel tlc rf 0.30 ( 1:2 hexanes  ethyl 
 acetate ) ; h nmr ( cdcl3 )  2.02 ( s , 3h ) , 
 2.04 ( s , 3h ) , 2.09 ( s , 3h ) , 2.10 ( s , 3h ) , 2.12 ( s , 3h ) , 2.15 ( s , 3h ) , 
 3.73 ( dd , 1h , j = 8.0 and 3.4 hz ) , 4.12 ( m , 3h ) , 
 4.34 ( m , 1h ) , 4.95 ( m , 5h ) , 5.10 ( m , 1h ) , 5.16 ( d , 1h , j = 3.3 hz ) , 5.25 ( m , 1h ) , and 5.37 ( m , 1h ) . to a solution of 
 25 mg ( 39 mol ) of 26 in 1
ml of dry acetonitrile 
 were added 7.0 mg ( 58 mol ) of 4-(n , n - dimethylamino)pyridine and a solution containing 12 mg 
 ( 58 mol ) of p - nitrophenyl chloroformate in 
 125 l of acetonitrile .
the reaction mixture was stirred at 
 room temperature for 1 h , at which time silica gel tlc analysis showed 
 complete consumption of the starting material .
the reaction mixture 
 was concentrated under diminished pressure , dissolved in 30 ml of 
 ethyl acetate , and then washed successively with 10 ml of h2o , 10 ml of 1 n aqueous hcl , and 10 ml of brine .
the ethyl acetate 
 layer was dried over na2so4 , filtered , and concentrated 
 under diminished pressure .
the residue was purified by flash chromatography 
 on a silica gel column ( 15  2 cm ) .
elution with 1:1  
 1:2 hexanes  ethyl acetate afforded the carbonate 27 as a colorless oil : yield 28 mg ( 92% ) ; silica gel tlc rf 0.38 ( 2:3 hexanes  ethyl acetate ) ; h nmr ( cdcl3 )  1.94 ( s , 3h ) , 2.06 ( s , 3h ) , 2.10 
 ( s , 3h ) , 2.15 ( s , 3h ) , 2.16 ( s , 3h ) , 2.21 ( s , 3h ) , 4.11 ( m , 6h ) , 4.41 
 ( m , 1h ) , 4.69 ( br s , 2h ) , 5.04 ( m , 2h ) , 5.16 ( m , 2h ) , 5.29 ( m , 1h ) , 
 5.48 ( m , 1h ) 5.83 ( d , 1h , j = 8.4 hz ) , 7.47 ( dd , 
 1h , j = 6.8 and 2 hz ) , and 8.31 ( dd , 1h , j = 6.8 and 2 hz ) ; c nmr ( cdcl3 ) 
  20.78 , 20.84 , 20.95 , 61.4 , 62.0 , 65.6 , 65.9 , 67.5 , 69.2 , 69.7 , 
 69.8 , 70.0 , 72.0 , 95.3 , 95.5 , 122.1 , 125.6 , 146.0 , 151.1 , 155.1 , 155.2 , 
 169.3 , 169.3 , 169.6 , 169.8 , 170.5 , and 170.7 ; mass spectrum ( esi ) m / z 825.1821 ( m + na ) ( c32h38n2o22na requires m / z 825.1808 ) . to a solution containing 
 1.0 mg ( 1.2 mol ) of 27 and 1.0 mg ( 0.9 mol ) 
 of cu(ii)deglycobleomycin a5 in 0.5 ml of anhydrous 
 dmf was added 5.0 l ( 3.7 mg ; 36 mol ) of dry triethylamine , 
 and
the reaction mixture was shaken at room temperature for 3 h. fifty 
 microliters ( 51 mg ; 1.6 mmol ) of hydrazine was added and the reaction 
 mixture was shaken at room temperature for 1 h and then concentrated 
 under diminished pressure .
ch3cn 
 and purified on an alltech alltima c18 reversed - phase semipreparative 
 ( 250  10 mm , 5 m ) hplc column by the use of 0.1% aqueous 
 cf3cooh and ch3cn mobile phases .
 ch3cn ) over a period of 35 min at a flow rate of 3 ml / min .
fractions 
 containing the desired product eluted at 17.0 min and were collected , 
 frozen , and lyophilized to give the cu(ii ) chelate of 6 as a light blue solid : yield 0.53 mg ( 39% ) .
following demetalation 
 with 15% aqueous edta , 0.20 mg ( 40% ) of 6 was isolated 
 as a colorless solid ; mass spectrum ( maldi - tof ) m / z 1484.9 ( m + h ) ( theoretical m / z 1484.6 ) ; mass spectrum ( esi ) m / z 742.7995 ( m + 2h ) ( c58h91n19o23s2 requires m / z 742.7983 ) .
two hundred nanograms 
 of supercoiled pbr322 
 or psp64 plasmid dna was treated with the appropriate concentrations 
 of freshly prepared fe [ from fe(nh4)2(so4)26h2o ] and ( deglyco)blm 
 solutions in a 15 l reaction mixture ( total volume ) containing 
 50 mm tris - hcl , ph 8.0 .
reactions were incubated at 25 c for 
 30 min and were then quenched by the addition of 3 l of native 
 gel loading buffer containing 30% glycerol and 0.125% ( w / v ) bromophenol 
 blue .
five microliters of the reaction mixture was loaded onto a 1% 
 agarose gel containing 9 mm tris 
borate buffer , ph 8.3 , and 
 320 m disodium ethylenediaminetetraacetate ( na2edta ) . 

the gel was run at 96 v for 2 h in a buffer solution containing 9 
 mm tris 
the gel was then stained in the running buffer solution supplemented 
 with 0.5 g / ml ethidium bromide for 1 h , followed by destaining 
 in water for 15 min .
du145 ( american 
 type culture collection ) human prostate cancer cells were grown in 
 log - phase mode in eagle s minimal essential medium ( mem ; gibco ) 
 supplemented with 10% fetal bovine serum and 1% penicillin  streptomycin . 

cells were trypsinized and seeded into a 96-well plate in 100 l 
 of culture medium at 5000 cells / well .
cells were incubated for 24 
 h , followed by removal of medium and replacement with medium containing 
 each bleomycin derivative at the appropriate concentration to the 
 wells .
the samples contained dimethyl sulfoxide ( dmso ) at a final 
 concentration of 5% .
following incubation with each compound for 24 
 h , 20 l of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 
 bromide ( mtt ) was added at a concentration of 5 mg / ml in phosphate - buffered 
 saline , ph 7.4 .
after incubation for 3 h at 37 c , the culture 
 medium was removed , and the crystals were dissolved in 200 l 
 of dmso . the 96-well plate was read in a multiwell plate reader with 
 an absorbance filter of 560 nm and a reference filter of 670 nm .
data 
 were analyzed by the use of qtiplot for determination of linear fit . 

sw480 colon 
 cancer cells ( atcc 
 ccl-228 ) were cultured in rpmi 1640 medium ( gibco , grand island , ny ) 
 supplemented with 10% fetal bovine serum ( hyclone , south logan , ut ) 
 and 1% penicillin 
du145 ( atcc htb-81 ) prostate cancer cells , pz - hpv-7 
 ( atcc crl-2221 ) normal prostate cells , wi-38 ( atcc ccl-75 ) normal 
 lung cells , and ccd-1105 kidtr ( atcc crl-2305 ) normal kidney cells 
 were grown in mem medium ( gibco , grand island , ny ) supplemented with 
 10% fetal bovine serum ( hyclone ) and 1% penicillin 
the cells were incubated at 37 c 
 under a humidified atmosphere of 5% co2 and 95% air .
fluorescence images were acquired 
 on a zeiss axiovert 200 m inverted microscope fitted with an axiocam 
 mrm camera equipped with a 300 w xenon lamp ( sutter , novato , ca ) and 
 cy5 and cy7 cyanine filter ( chroma , bellows falls , vt ) .
the cells 
 were grown on 16-well lab - tek glass chamber slides at a cell density 
 of 5000 cells / well ( thermo scientific , waltham , ma ) at 37 c 
 for 48 h. cells were rinsed twice with phosphate - buffered saline ( pbs ) 
 and the medium was replaced with rpmi 1640 ( no phenol red ) when the 
 cell density reached 70% confluence .
cells were incubated 
 at 37 c for 1 h , washed with pbs , and then fixed with 4% paraformaldehyde 
 at 37 c for 5 min .
finally , the slide was mounted with prolong 
 antifade gold reagent with dapi ( invitrogen ) .
all images were recorded 
 and the target cells were counted by use of a 40 oil objective . 
 to ensure accurate intensity measurements , the exposure time and laser 
 time were kept identical .
pixel intensity was quantified by the use 
 of axiovision release 4.7 version software , and the mean pixel intensity 
 was generated as gray level .
 the disaccharide moiety is responsible 
 for the tumor cell targeting 
 properties of bleomycin ( blm ) . 
 while the aglycon ( deglycobleomycin ) 
 mediates dna cleavage in much the same fashion as bleomycin , it exhibits 
 diminished cytotoxicity in comparison to blm . 
 these findings suggested 
 that blm might be modular in nature , composed of tumor - seeking and 
 tumoricidal domains . to explore this possibility , 
 blm analogues were 
 prepared in which the disaccharide moiety was attached to deglycobleomycin 
 at novel positions , namely , via the threonine moiety or c - terminal 
 substituent . 
 the analogues were compared with blm and deglycoblm for 
 dna cleavage , cancer cell uptake , and cytotoxic activity . 
 blm is more 
 potent than deglycoblm in supercoiled plasmid dna relaxation , while 
 the analogue having the disaccharide on threonine was less active 
 than deglycoblm and the analogue containing the c - terminal disaccharide 
 was slightly more potent . while having unexceptional dna cleavage 
 potencies , both glycosylated analogues were more cytotoxic to cultured 
 du145 prostate cancer cells than deglycoblm . 
 dye - labeled conjugates 
 of the cytotoxic blm aglycons were used in imaging experiments to 
 determine the extent of cell uptake . 
 the rank order of internalization 
 efficiencies was the same as their order of cytotoxicities toward 
 du145 cells . 
 these findings establish a role for the blm disaccharide 
 in tumor targeting / uptake and suggest that the disaccharide moiety 
 may be capable of delivering other cytotoxins to cancer cells . while 
 the mechanism responsible for uptake of the blm disaccharide selectively 
 by tumor cells has not yet been established , data are presented which 
 suggest that the metabolic shift to glycolysis in cancer cells may 
 provide the vehicle for selective internalization . 
Introduction
Results
Discussion
Conclusions
Experimental
Procedures