melanocortin 1 ( mc1 ) receptor is an attractive 
 molecular target for melanoma imaging because of its overexpression 
 on both murine and human melanoma cells .
recently , we have identified a class of tc - labeled 
 -melanocyte stimulating hormone ( -msh ) peptides to target 
 mc1 receptors for melanoma imaging .
specifically , the cyclic rxd motifs 
 { arg - x - asp - dtyr - asp , x = gly , ala , val , thr , ser , nle , phe , 
 and dphe } were attached to [ cys , d - phe , arg]-msh313 via a lysine linker to yield rxd - lys-(arg)ccmsh peptides . 

interestingly , single amino acid at the x position yielded a profound 
 impact on the melanoma targeting and clearance properties of tc - rxd - lys-(arg)ccmsh peptides .
for instance , 
 the substitution of gly in tc - rgd - lys-(arg)ccmsh with ala , thr , val , and ser improved the mc1 receptor binding 
 affinities and enhanced the melanoma uptake in b16/f1 melanoma - bearing 
 c57 mice . on the other hand
, the substitution of gly in tc - rgd - lys-(arg)ccmsh with nle decreased the 
 mc1 receptor binding affinity .
although the substitution of gly in tc - rgd - lys-(arg)ccmsh with phe and dphe increased the mc1 receptor binding affinities , both tc - rfd - lys-(arg)ccmsh and tc - rfd - lys-(arg)ccmsh exhibited much higher liver uptake as compared to tc - rgd - lys-(arg)ccmsh . despite the promising melanoma targeting results , extremely 
 high renal uptake ( 67135% id / g at 2 h postinjection ) is a 
 common issue associated with tc - rxd - lys-(arg)ccmsh peptides .
thus , it is desirable to reduce the nonspecific renal 
 uptake of tc - rxd - lys-(arg)ccmsh peptides 
 to facilitate their potential therapeutic applications . in our previous 
 reports ,
l - lysine co - injection significantly reduced the 
 renal uptake of tc - rxd - lys-(arg)ccmsh peptides 
 by 37%51% at 2 h postinjection without affecting their tumor 
 uptake.l - lysine is a positively charged amino acid . 

the effect of l - lysine co - injection in reducing the renal 
 uptake indicated that the overall positive charges of tc - rxd - lys-(arg)ccmsh peptides contributed to their 
 nonspecific renal uptake .
obviously , the substitution of the positively 
 charged lys linker with a neutral amino acid can decrease the overall 
 charges of tc - rxd - lys-(arg)ccmsh peptides . 
 according to the effect of l - lysine co - injection in decreasing 
 the renal uptake
, we hypothesized that the substitution of the lys 
 linker with a neutral -ala linker would decrease the renal 
 uptake of tc - rxd - lys-(arg)ccmsh peptides . 
 to examine our hypothesis , we synthesized six peptides with -ala 
 linkers , namely , peptides 16 .
the 
 mc1 receptor binding affinities of these six peptides were examined 
 in b16/f1 melanoma cells .
we further 
 determined the biodistribution properties in b16/f1 melanoma - bearing 
 c57 mice for these six tc - peptides .
thereafter , we determined 
 the imaging property of tc-4 in b16/f1 
 melanoma - bearing c57 mice .
the peptides were synthesized and purified by reverse phase - high 
 performance liquid chromatography ( rp - hplc ) according to our previously 
 published procedures .
the chemical purities of 
 the peptides were greater than 95% after the hplc purification ( table 1 ) .
the measured molecular weight was 2123 da for peptide 1 , 2137 da for peptide 2 , 2135 da for peptide 3 , 2107 da for peptide 4 , 2064 da for peptide 5 , and 2080 da for peptide 6 ( table 1 ) .
the ic50 value was 2.76  0.51 nm for peptide 1 , 1.56  0.63 nm for peptide 2 , 1.99  
 0.16 nm for peptide 3 , 0.35  0.01 nm for peptide 4 , 3.34  0.28 nm for peptide 5 , and 3.84 
  0.71 nm for peptide 6 in b16/f1 melanoma cells , 
 respectively .
competitive binding curves of peptide 1 (  , pink ) , peptide 2 (  , black ) , 
 peptide 3 (  , orange ) , peptide 4 (  , 
 blue ) , peptide 5 (  , green ) , and peptide 6 (  , red ) in b16/f1 murine melanoma cells .
the ic50 values were 2.76  0.51 nm for peptide 1 , 1.56  0.63 nm for peptide 2 , 1.99  0.16 
 nm for peptide 3 , 0.35  0.01 nm for peptide 4 , 3.34  0.28 nm for peptide 5 , and 3.84 
  0.71 nm for peptide 6 .
the tc - peptides were separated from their excess nonlabeled peptides 
 by rp - hplc .
the specific activities of tc-1 , tc-2 , tc-3 , tc-4 , tc-5 , and tc-6 were 8.62  10 , 8.57  10 , 8.57  10 , 8.68  
 10 , 8.85  10 , 8.79  10 mbq / g , respectively .
the retention times of tc-1 , tc-2 , tc-3 , tc-4 , tc-5 , and tc-6 were 13.1 , 13.0 , 14.1 , 
 13.7 , 17.0 , and 14.2 min , respectively .
all six tc - peptides 
 were stable in mouse serum at 37 c for 24 h ( figure 3 ) .
radioactive hplc profiles of tc-1 ( a ) , tc-2 ( b ) , tc-3 ( c ) , tc-4 ( d ) , tc-5 ( e ) , and tc-6 ( f ) in 
 mouse serum after incubation at 37 c for 24 h. the arrows denote 
 the original retention times of tc-1 ( 13.1 
 min ) , tc-2 ( 13.0 min ) , tc-3 ( 14.1 min ) , tc-4 ( 13.7 min ) , tc-5 ( 17.0 min ) , and tc-6 ( 14.2 min ) , prior to the incubation in mouse serum .
the melanoma targeting and pharmacokinetic 
 properties of tc-1 , tc-2 , tc-3 , tc-4 , tc-5 , and tc-6 are shown in tables 27 .
all six tc - peptides exhibited similar 
 tumor uptake pattern in b16/f1 melanoma - bearing c57 mice .
the highest 
 tumor uptake appeared either at 2 or 4 h postinjection . among these 
 six tc - peptides ,
tc-4 showed 
 the highest tumor uptake of 15.66  6.19% id / g at 2 h postinjection . 

the tumor uptake of tc-4 gradually decreased 
 to 14.67  3.81 and 7.79  2.68% id / g at 4 and 24 h postinjection . 

co - injection of 10 g ( 6.1 nm ) of nonradiolabeled ndp - msh with tc-4 decreased the tumor uptake to 2.43  
 0.53% id / g at 2 h postinjection , demonstrating that the tumor uptake 
 was mc1 receptor - mediated .
kidneys were the excretion routes 
 for all six tc - peptides . among these six tc - peptides ,
tc-4 showed the lowest renal 
 uptake of 20.18  3.86% id / g at 2 h postinjection .
the renal 
 uptake of tc-4 gradually decreased to 19.83 
  6.34 and 3.92  0.99% id / g at 4 and 24 h postinjection . 

co - injection of 10 g ( 6.1 nm ) of nonradiolabeled ndp - msh with tc-4 did not significantly reduce the renal 
 uptake ( p > 0.05 ) at 2 h postinjection , indicating 
 that the renal uptake was nonspecific .
the substitution of the positively 
 charged lys linker with the neutral -ala dramatically decreased 
 the renal uptake of tc - rxd--ala-(arg)ccmsh peptides .
interestingly , further reduction of 
 the overall positive charges of tc-5 and tc-6 did not decrease the renal uptake further 
 as compared to tc-4 .
approximately 6878% 
 of tc - peptides cleared through the urinary system by 
 2 h postinjection , whereas approximately 7786% of tc - peptides washed out through the urinary system by 4 h postinjection . 

the effect of l - lysine co - injection on the renal uptake of tc-4 at 2 h postinjection is presented in figure 4 .
co - injection of 15 mg of l - lysine significantly 
 ( p < 0.05 ) reduced the renal uptake of tc-4 from 20.18  3.86% id / g to 13.06  3.62% 
 id / g without significantly affecting the tumor uptake at 2 h postinjection .
the data are presented as percent injected dose / gram or as percent 
 injected dose ( mean  sd , n = 4 ) .
(  ) p < 0.05 
 for determining the significance of differences in tumor and kidney 
 uptake between tc-1 with or without ndp - msh 
 peptide blockade at 2 h postinjection .
the data are presented as percent 
 injected dose / gram or as percent injected dose ( mean  sd , n = 4 ) .
(  ) p < 0.05 for determining the significance of differences 
 in tumor and kidney uptake between tc-2 with or without ndp - msh peptide blockade at 2 h postinjection .
the data are presented as percent injected dose / gram or as percent 
 injected dose ( mean  sd , n = 4 ) .
(  ) p < 0.05 
 for determining the significance of differences in tumor and kidney 
 uptake between tc-3 with or without ndp - msh 
 peptide blockade at 2 h postinjection . the data are presented as percent injected dose / gram or as percent 
 injected dose ( mean  sd , n = 4 ) .
(  ) p < 0.05 
 for determining the significance of differences in tumor and kidney 
 uptake between tc-4 with or without ndp - msh 
 peptide blockade at 2 h postinjection . the data are presented as percent injected dose / gram or as percent 
 injected dose ( mean  sd , n = 4 ) .
(  ) p < 0.05 
 for determining the significance of differences in tumor and kidney 
 uptake between tc-5 with or without ndp - msh 
 peptide blockade at 2 h postinjection . the data are presented as percent injected dose / gram or as percent 
 injected dose ( mean  sd , n = 4 ) .
(  ) p < 0.05 
 for determining the significance of differences in tumor and kidney 
 uptake between tc-6 with or without ndp - msh 
 peptide blockade at 2 h postinjection .
effect of l - lysine co - injection on the tumor and kidney 
 uptake of tc-4 at 2 h postinjection .
the 
 blue and green columns represent the tumor and renal uptake of tc-4 without and with l - lysine co - injection : 
 (  ) p < 0.05 for determining the significance 
 of differences in tumor and kidney uptake between tc-4 without and with l - lysine co - injection . because tc-4 showed 
 the highest tumor uptake and the lowest renal uptake than the other 
 five tc - peptides at 2 h postinjection , we further determined 
 the tumor imaging property , specificity of tumor uptake , and urinary 
 metabolites of tc-4 in b16/f1 melanoma - bearing 
 c57 mice .
whole - body spect / ct image at 2 h postinjection is presented 
 in figure 5 .
flank b16/f1 melanoma lesions 
 were clearly visualized by spect using tc-4 as an imaging probe .
the spect image of tumor accurately matched 
 its anatomical location obtained in the ct image .
the spect image 
 showed high contrast of tumor to normal organ except for kidneys , 
 which was consistent with the biodistribution results .
as shown in 
 figure 5 , the tumor uptake was blocked by unlabeled 
 ndp - msh , demonstrating that the tumor uptake was receptor - mediated . 

the urinary metabolites of tc-4 at 2 h 
 postinjection are shown in figure 6 .
representative 
 whole - body spect / ct image of b16/f1 melanoma - bearing c57 mice 2 h 
 after injection of tc-4 without ( a ) and 
 with ( b ) peptide blockade .
flank melanoma lesions ( t ) are highlighted 
 with an arrow on the image .
the arrow denotes the original 
 retention time of tc-4 prior to tail vein 
 injection .
in our previous reports , we have found the importance of single amino acid at the x position 
 in the tumor targeting properties of tc - rxd - lys-(arg)ccmsh peptides in b16/f1 melanoma - bearing c57 mice . specifically , 
 the substitution of gly in tc - rgd - lys-(arg)ccmsh with ala , thr , val , and ser improved the mc1 receptor binding 
 affinities and enhanced the melanoma uptake in b16/f1 melanoma - bearing 
 c57 mice . despite the promising melanoma targeting results 
 associated with tc - rxd - lys-(arg)ccmsh peptides , 
 it is desirable to reduce the nonspecific renal uptake ( 67135% 
 id / g at 2 h postinjection ) of tc - rxd - lys-(arg)ccmsh peptides to facilitate their potential therapeutic applications . 
 in this study
, we substituted the positively charged lys linker with 
 the neutral -ala linker to determine whether such linker change 
 could reduce the renal uptake of tc - rxd--ala-(arg)ccmsh peptides .
furthermore , we replaced the rad moiety 
 with nad and ead moieties to examine whether the further reduction 
 of the overall positive changes of tc - nad--ala-(arg)ccmsh and tc - ead--ala-(arg)ccmsh could decrease their renal uptake further . the substitution 
 of lys linker with -ala linker slightly affected the receptor 
 binding affinities of rxd--ala-(arg)ccmsh peptides . 

the receptor binding affinities of rxd--ala-(arg)ccmsh peptides were approximately 2-fold weaker than rxd - lys-(arg)ccmsh peptides , respectively . despite the fact that the 
 -his - dphe - arg - trp- motif is the binding moiety for the mc1 
 receptor , the decrease in receptor binding affinity with the -ala 
 linker substitution indicated that the linker might somehow interact 
 with the receptor binding moiety .
the decrease in receptor binding 
 affinities of rxd--ala-(arg)ccmsh peptides also 
 resulted in the reduction in tumor uptake of tc - rxd--ala-(arg)ccmsh peptides by 2145% in b16/f1 melanoma - bearing 
 c57 mice .
specifically , the tumor uptake of tc-4 was 79% of the tumor uptake of tc - rad - lys-(arg)ccmsh at 2 h postinjection .
the substitution of lys linker with -ala linker dramatically 
 decreased the renal uptake of tc - rxd--ala-(arg)ccmsh peptides by 6479% in b16/f1 melanoma - bearing 
 c57 mice .
for instance , the renal uptake of tc-4 was only 22% of the renal uptake of tc - rad - lys-(arg)ccmsh at 2 h postinjection .
it is worthwhile to note that there is a positively charged arg 
 residue in the rad moiety of tc-4 .
thus , 
 we were interested in whether the replacement of arg with nle ( neutral ) 
 and glu ( negatively charged ) could further decrease the renal uptake 
 of tc-5 and tc-6 as compared to tc-4 .
interestingly , the 
 replacement of arg with nle and glu did not further decrease the renal 
 uptake of tc-5 and tc-6 as compared to tc-4 .
clearly , 
 the tumor targeting and clearance properties of tc-4 were more favorable than the other tc - peptides 
 investigated in this study .
. the b16/f1 melanoma lesions 
 could be clearly visualized by spect / ct using tc-4 as an imaging probe . at the present time
, tc-(arg)ccmsh and tc(edda)-hynic - ggnle - cycmshhex were reported as promising cyclic imaging probes for melanoma .
remarkably , tc-4 displayed comparably 
 high melanoma uptake ( 14.67  3.81% id / g ) as tc-(arg)ccmsh and tc(edda)-hynic - ggnle - cycmshhex at 4 h postinjection .
however , the renal uptake of tc-4 was higher than those of tc-(arg)ccmsh ( 11.66  1.44% id / g ) and tc(edda)-hynic - ggnle - cycmshhex ( 7.52  0.96% id / g ) . 

the difference in renal uptake was likely due to the structural differences 
 among these tc - peptides .
interestingly , we found that l - lysine co - injection significantly reduced the renal uptake 
 of tc-4 by 35% at 2 h postinjection without 
 affecting its tumor uptake significantly ( figure 4 ) .
therefore , l - lysine co - injection could be utilized 
 to further decrease the renal uptake of re - rad - lys-(arg)ccmsh to facilitate its potential therapeutic application .
the substitution of the lys linker with the -ala linker 
 dramatically decreased the renal uptake of tc - rxd--ala-(arg)ccmsh peptides . among these six tc - peptides ,
tc-4 exhibited the highest tumor uptake and 
 the lowest renal uptake at 2 h postinjection .
the replacement of arg 
 with nle and glu did not further decrease the renal uptake of tc-5 and tc-6 as 
 compared to tc-4 .
the tumor targeting and 
 clearance properties of tc-4 highlighted 
 it as a lead peptide for future studies .
i - tyr-[nle , dphe]--msh { i-(tyr)-ndp - msh } 
 was obtained from perkinelmer , inc .
all other chemicals used in 
 this study were purchased from thermo fischer scientific ( waltham , 
 ma ) and used without further purification .
b16/f1 murine melanoma 
 cells were obtained from american type culture collection ( manassas , 
 va ) .
six new peptides were synthesized using fluorenylmethyloxycarbonyl 
 ( fmoc ) chemistry according to our previously published procedures with slight modification on sieber amide resin by an advanced chemtech 
 multiple - peptide synthesizer ( louisville , ky ) .
briefly , 70 mol 
 of sieber amide resin and 210 mol of fmoc - protected amino acids 
 were used for the synthesis .
intermediate scaffolds of h2n - arg(pbf)-ser / thr / val - asp(otbu)-dtyr(tbu)-asp(o-2-phenylisopropyl)--ala - cys(trt)-cys(trt)-glu(otbu)-his(trt)-dphe - arg(pbf)-trp(boc)-cys(trt)-arg(pbf)-pro - val 
 and h2n - arg(pbf)/nle / glu(otbu)/-ala - asp(otbu)-dtyr(tbu)-asp(o-2-phenylisopropyl)--ala - cys(trt)-cys(trt)-glu(otbu)-his(trt)-dphe - arg(pbf)-trp(boc)-cys(trt)-arg(pbf)-pro - val 
 were synthesized on sieber amide resin .
the protecting group of 2-phenylisopropyl 
 of each scaffold was removed , and each peptide was cleaved from the 
 resin treating with a mixture of 2.5% of trifluoroacetic acid ( tfa ) 
 and 5% of triisopropylsilane .
after the precipitation with ice - cold 
 ether and characterization by ms , each protected peptide was dissolved 
 in h2o / ch3cn ( 50:50 ) and lyophilized to remove 
 the reagents such as tfa and triisopropylsilane .
each protected peptide 
 was further cyclized by coupling the carboxylic group from the asp 
 with the  amino group from the arg , nle , or glu at the n - terminus . 

the cyclization reaction was achieved by overnight reaction in dimethylformamide 
 ( dmf ) using benzotriazole-1-yl - oxy - tris - pyrrolidinophosphonium hexafluorophosphate 
 ( pybop ) as a coupling agent in the presence of n , n - diisopropylethylamine ( dipea ) .
the protecting groups were 
 totally removed by treating with a mixture of tfa , thioanisole , phenol , 
 water , ethanedithiol , and triisopropylsilane ( 87.5:2.5:2.5:2.5:2.5:2.5 ) 
 for 2 h at room temperature ( 25 c ) .
each peptide was precipitated 
 and washed with ice - cold ether four times , purified by rp - hplc , and 
 characterized by liquid chromatography  mass spectrometry ( lc  ms ) . 

the chemical purity of each peptide was determined by waters rp - hplc 
 instrument ( milford , ma ) on a grace vydac c-18 reverse phase analytic 
 column ( deerfield , il ) using a 20 min gradient of 1828% acetonitrile 
 in 20 mm hcl aqueous solution at a flow rate of 1 ml / min .
the ic50 values of the peptides for the mc1 receptor were determined in b16/f1 
 melanoma cells .
the b16/f1 cells were seeded into 
 a 24-well cell culture plate at a density of 2.5  10 cells / well and incubated at 37 c overnight .
after being washed 
 with binding medium { modified eagle s medium with 25 mm n-(2-hydroxyethyl)piperazine - n-(2-ethanesulfonic 
 acid ) ( hepes ) , ph 7.4 , 0.2% bovine serum albumin ( bsa ) , 0.3 mm 1,10-phenathroline } , 
 the cells were incubated at 25 c for 2 h with approximately 
 30  000 counts per minute ( cpm ) of i-(tyr)-ndp - msh in the presence of increasing concentrations ( 1010 m ) of each peptide 
 in 0.3 ml of binding medium .
the cells were rinsed twice with 0.5 ml of ice - cold , 
 ph 7.4 , 0.2% bsa/0.01 m phosphate buffered saline ( pbs ) to remove 
 any unbound radioactivity and lysed in 0.5 ml of 1 m naoh for 5 min . 

the activities associated with the cells were measured in a wallac 
 2480 automated  counter ( perkinelmer , nj ) .
the ic50 value for each peptide was calculated using prism software ( graphpad 
 software , la jolla , ca ) .
the standard deviation of ic50 value was generated by two independent experiments in triplicate 
 for each peptide .
the peptides 
 were labeled with tc via a direct reduction reaction 
 using sncl2 as a reducing agent .
briefly , 10 l of 
 1 mg / ml sncl2 in 0.1 m hcl , 40 l of 0.5 m nh4oac ( ph 5.2 ) , 100 l of 0.2 m na2 tartate 
 ( ph 9.2 ) , 100 l of fresh tco4 solution ( 3774 mbq ) , and 10 l of 1 
 mg / ml of each peptide in aqueous solution were added into a reaction 
 vial and incubated at 25 c for 20 min to form the tc - labeled peptide .
each tc - peptide was purified to 
 a single species by waters rp - hplc ( milford , ma ) on a grace vydac 
 c-18 reverse phase analytic column ( deerfield , il ) using a 20 min 
 gradient of 1828% acetonitrile in 20 mm hcl aqueous solution 
 at a flow rate of 1 ml / min .
each purified peptide was purged with 
 n2 gas for 20 min to remove the acetonitrile .
the ph of 
 final peptide solution was adjusted to 7.4 with 0.1 n naoh and sterile 
 normal saline for stability , biodistribution , and imaging studies . 

the serum stabilities of tc-1 , tc-2 , tc-3 , tc-4 , tc-5 , and tc-6 were determined by incubation in mouse serum at 
 37 c for 24 h and monitored for degradation by rp - hplc .
briefly , 
 100 l of each hplc - purified peptide solution ( 7.4 mbq ) 
 was added into 100 l of mouse serum ( sigma - aldrich corp , st . 

louis , mo ) and incubated at 37 c for 24 h. after the incubation , 
 200 l of a mixture of ethanol and acetonitrile ( v : v = 1:1 ) 
 was added to precipitate the serum proteins .
the resulting mixture 
 was centrifuged at 16000 g for 5 min to collect the 
 supernatant .
the supernatant was purged with n2 gas for 
 30 min to remove the ethanol and acetonitrile .
the resulting sample 
 was mixed with 500 l of water and injected into rp - hplc for 
 analysis using the gradient described above .
all the animal studies were conducted in compliance with institutional 
 animal care and use committee approval ( 12 - 100851-hsc ) .
the biodistribution 
 properties of the tc - peptides were determined in b16/f1 
 melanoma - bearing c57 female mice ( harlan , indianapolis , in ) .
each 
 c57 mouse was subcutaneously inoculated on the right flank with 1 
  10 b16/f1 cells .
the weight of tumors reached approximately 
 0.2 g at 10 days after cell inoculation .
each melanoma - bearing mouse 
 was injected with 0.037 mbq of each tc - peptide via the 
 tail vein .
groups of four mice were sacrificed at 0.5 , 2 , 4 , and 24 
 h postinjection , and tumors and organs of interest were harvested , 
 weighed , and counted .
the specificity of tumor uptake was determined by co - injecting 
 each tc - peptide with 10 g ( 6.1 nmol ) of unlabeled 
 ndp - msh at 2 h postinjection .
the effect of l - lysine 
 co - injection on the renal uptake of tc-4 was examined in b16/f1 melanoma - bearing c57 .
a group of four mice 
 were injected with an aqueous mixture of 0.037 mbq of tc-4 and 15 mg of l - lysine .
the mice were sacrificed 
 at 2 h postinjection , and tumor and kidneys were harvested , weighed , 
 and counted .
tc-4 was the lead peptide 
 because of its highest tumor uptake and the lowest renal uptake .
thus , 
 we further determined the melanoma imaging property of tc-4 and the specificity of melanoma uptake .
approximately 
 3.74.1 mbq of tc-4 with or without 
 10 g ( 6.1 nmol ) of unlabeled ndp - msh was injected into b16/f1 
 melanoma - bearing c57 mice via the tail vein , respectively .
the mice 
 were euthanized for small animal spect / ct ( nano - spect / ct , bioscan , 
 washington , dc ) imaging 2 h postinjection .
the 9 min ct imaging was 
 immediately followed by the spect imaging of whole body .
reconstructed data from spect 
 and ct were visualized and co - registered using invivoscope ( bioscan , 
 washington , dc ) .
approximately 3.7 mbq of tc-4 was injected into a b16/f1 melanoma - bearing c57 
 mouse via the tail vein to determine the urinary metabolites .
the collected 
 urine sample was centrifuged at 16000 g for 5 min 
 before the hplc analysis .
a 20 min gradient of 1626% acetonitrile/20 
 mm hcl with a flow rate of 1 ml / min was used for urine analysis .
statistical analysis was performed 
 using the student s t test for unpaired data 
 to determine the significance of differences in tumor and kidney uptake 
 with / without peptide blockade in biodistribution studies described 
 above .
 the 
 purpose of this study was to examine whether the substitution of the 
 lys linker with the -ala could reduce the renal uptake of 99mtc - labeled arg - x - asp - conjugated and x - ala - asp - conjugated 
 -melanocyte stimulating hormone ( -msh ) peptides . 
 rsd--ala-(arg11)ccmsh ( 1 ) { c[arg - ser - asp - dtyr - asp]--ala - cys - cys - glu - his - dphe - arg - trp - cys - arg - pro - val - nh2 } , rtd--ala-(arg11)ccmsh ( 2 ) , rvd--ala-(arg11)ccmsh ( 3 ) , rad--ala-(arg11)ccmsh 
 ( 4 ) , nad--ala-(arg11)ccmsh ( 5 ) , and ead--ala-(arg11)ccmsh ( 6 ) peptides 
 were synthesized and evaluated for their melanocortin 1 ( mc1 ) receptor 
 binding affinities in b16/f1 melanoma cells . 
 the biodistribution of 
 their 99mtc - conjugates were determined in b16/f1 melanoma - bearing 
 c57 mice . 
 the substitution of the lys linker with -ala linker 
 dramatically reduced the renal uptake of all six 99mtc - peptides . 
 99mtc-4 exhibited the highest melanoma uptake 
 ( 15.66  6.19% id / g ) and the lowest kidney uptake ( 20.18  
 3.86% id / g ) among these 99mtc - peptides at 2 h postinjection . 
 
 the b16/f1 melanoma lesions could be clearly visualized by single 
 photon emission computed tomography ( spect)/ct using 99mtc-4 as an imaging probe . 
Introduction
Results
Discussion
Conclusions
Experimental
Section