it is estimated that 
 there are 1.5 billion people worldwide suffering 
 at any given time from some type of central nervous system ( cns ) disorder . novel cns drugs have the potential to further improve quality of life 
 and to reduce the disease burden for these serious diseases and disorders . since the discovery of the two endogenous pentapeptides , 
 met - enkephalin and leu - enkephalin , in 1975 , more than 250 endogenous 
 neuropeptides have been identified , and there is now a broad vista 
 for the application of these peptides in pharmacology , especially 
 for the opioids that are so widespread throughout the cns . now in the fourth decade of research in this 
 area , many potent and selective peptide agonists have been developed 
 for the three cloned opioid receptors , but some crucial drawbacks 
 still persist that dampen enthusiasm for the use of these compounds 
 as peptide - based drugs , primarily instability in vivo and poor blood  brain 
 barrier ( bbb ) penetration . numerous methods have been devised 
 and successfully applied to 
 overcome metabolic instability and high clearance of peptides . the bbb a component of the 
 neurovascular unit , a structure consisting of endothelial cells of 
 brain and spinal cord capillaries , astrocytes , basement membrane , 
 pericytes , and neurons in physical proximity to the endothelium that 
 varies in composition and function from site to site within the brain . it is known that the bbb has anatomic and neuroprotective 
 functions because of the presence of oxidative enzymes and peptidases 
 such as aminopeptidase , arylamidase , and enkephalinase . the ability of drugs to diffuse passively 
 across the bbb has been predicted by molecular size , charge , hydrogen 
 bonding , and lipid solubility , but it is not clear how applicable 
 lipinski s rules are to peptides . several modifications have been studied in an effort 
 to overcome the bbb penetration problem , including lipidization , structural modification to enhance stability , glycosylation , nutrient 
 transporters , prodrugs , vector - based trojan horses , cationization , and conjugation to or 
 encapsulation by polymers . glycosylation 
 has been shown to improve antinociceptive potency and bioavailability 
 of glycopeptides via higher metabolic stability , reduced clearance , and improved 
 bbb transport . some bbb penetration studies 
 with glycopeptide agonists related to enkephalins have shown up to 
 a 3-fold increase in the rate of brain delivery of these analogues 
 compared with the unglycosylated parent peptides . recent studies with glycopeptides in micelles indicate 
 that amphipathicity of the glycopeptides is an important factor in 
 bbb penetration . it has been suggested 
 that glycosylation can alter tissue distribution patterns of glycopeptide 
 drugs and affect interactions with receptors . the endogenous 
 opioid -endorphin has the met - enkephalin peptide sequence yggfm 
 at the n - terminus and consists of 31 residues . kaiser and co - workers synthesized several -endorphins with 
 the met - enkephalin sequence , a hydrophilic linking segment , s--amino--hydroxymethylbutyric 
 acid ( home - gaba ) , replacing residues 612 , and an amphiphilic 
 helical segment between the helix breaker residues pro(13 ) and gly(30 ) . the circular dichroism ( cd ) spectra of all mimics , with minimal homology 
 to the -endorphin sequence , showed minima at 210 and 222 nm , 
 indicative of -helical structure . compared to -endorphin , the peptide mimics were 2 to 3 times 
 more potent in - and -opioid receptor binding assays , 
 about equipotent in the -receptor binding assay , and possessed 
 strong resistance toward proteolytic enzymes . these findings strongly suggested that the amphipathic -helical 
 structure in the c - terminal region of -endorphin plays a key 
 role in receptor binding and opioid activity as well as resistance 
 to proteolysis of mimic analogues . kyle and co - workers designed and 
 synthesized several conformationally constrained nociceptin ( nc / orl-1 ) 
 analogues , where they exploited the -helical - promoting 
 residues -aminoisobutyric acid ( aib ) and n - methyl alanine ( meala ) as replacement(s ) for ala7 , ala11 , or ala15 in the native nc c - terminal sequence . the importance of -helical address segments of peptides has 
 also been demonstrated in the secretin family of peptide receptors , 
 including crf , secretin , and vip . the interaction of pacap138 with a phospholipid membrane has been shown to be involved in binding 
 and receptor specificity , increasing peptide stability , and amplifying 
 bioactivity in vivo . on the basis of the 
 evidence that glycosylation decreases lipophilicity and on the hypothesis 
 that amphipathic properties of the helix could help guide a membrane - associated 
 peptide to its specific receptor , three generations of glycosylated 
 -endorphin analogues have now been synthesized for study : the first bearing longer helices too stable to engage in biousian behavior , the second being this study , and the third bearing more flexible linkages between the 
 opioid message and the helix address , which will be published shortly . it is of interest that some of the longer endorphin glycopeptides 
 analogues penetrated the bbb at higher rates than the shorter enkephalin 
 glycopeptide analogues . the influence 
 of the amphipathic helix in tandem with glycosylation on drug delivery 
 is therefore of great interest and was further examined using a series 
 of -endorphin glycopeptide analogues of varying helicity and 
 bearing different sugar moieties . to understand the conformation and 
 dynamics of membrane - bound peptides or integral proteins , several 
 model systems have been developed to mimic features of the membrane . it is generally accepted that the detergent sodium dodecyl sulfate 
 ( sds ) mimics the membrane - like environment and therefore its use has 
 been reported in the literature extensively to study peptide schwyzer s membrane compartment 
 concept suggests that amphipathic helices will promote binding with the receptors via 
 a 2d search of the membrane rather than a 3d search of the aqueous 
 compartment ( cf . reduction of dimensionality . 
 the calculated hydrophilic ( red ) and 
 hydrophobic ( blue ) connolly surfaces are illustrated for glycopeptide 
 l1 in side view ( a ) and down the axis of the helical address region 
 ( b ) . the 9th and 12th residues were replaced by -aminoisobutyric 
 acid ( aib ) , alanine , or glycine to adjust the helicity by increments . ( c ) the sugar can play an important role in drug transport by pulling 
 the glycopeptide away from membranes into the aqueous milieu to enable 
 membrane hopping . the amphipathic helix promotes 
 2d searching of the membrane in order to facilitate receptor binding . according to detailed analysis 
 of globular proteins physical - chemical 
 and structural properties , segrest and co - workers grouped amphipathic 
 helices into different classes ( a , h , l , g , k , c , and m ) according to the geometric distribution of lipophilic 
 and hydrophilic residues . class h , polypeptide hormones , typically 
 contain two distinct functional domains , a short n - terminal domain 
 including a specific message segment that binds to 
 the transmembrane portion of the receptor and a much longer amphipathic 
 helix , or address segment , located at the c - terminal 
 portion of the peptide . the helical domain may provide enhanced receptor 
 targeting in a relatively nonspecific manner by increasing affinity 
 for the hydrophobic membrane that contains the receptor . lipid interaction leading to cell penetration 
 plays a major role in their activity by one of two general mechanisms : 
 ( 1 ) transmembrane pore formation via a barrel - stave mechanism and 
 ( 2 ) membrane destruction / solubilization via a carpet mechanism . it is proposed that exploitation of the first 
 step of these mechanisms by class h glycopeptides will allow for reversible 
 adsorption to biological membranes without membrane disruption . this 
 putative glycopeptide lipid interaction allows peptides with 
 amphipathic helix conformation to float in the cell membrane , exposing 
 the hydrophobic side to the hydrophobic membrane and the hydrophilic 
 side to the aqueous exterior of the cell . furthermore , it is proposed 
 that this transient interaction with biological membranes is essential 
 for crossing cellular barriers ( not membranes ) , such as the endothelial 
 layer of cells that compose an important part of the bbb . the 
 peptides and glycopeptides examined in the current studies 
 were designed in accordance with kaiser s classic studies of 
 -endorphin combined with a simple edmundson wheel approach to introduce amphipathicity 
 per segrest s insights . molecular 
 mechanics calculations confirmed the potential for helical amphipathic 
 structures for the glycopeptides . in this study , all of the glycopeptides 
 shared the same message segment , ytgfl , used in previously 
 published studies . the helix - breaking 
 residue pro was used to link the n - terminal 
 message domain and c - terminal helix . the three series ( table 1 ) bore either an unglycosylated l - serine 
 residue ( u series ) , an l - serine monosaccharide 
 bearing a -o - glucose ( g series ) , 
 or an l - serine disaccharide bearing a -o - lactose ( l series ) . each series of seven ligands differed 
 in the address domain sequence residues at position 9 or 12 , where 
 aib , ala , and gly were introduced into the c - terminal address to obtain 
 different helix propensities . all of the helical 
 conformations were presumably stabilized by a salt bridge between 
 glu10 and lys14 ( i  i + 4 ) . s = l - serine , 
 s * = -o - glucosyl - l - serine , s * * = 
 -o - lactosyl - l - serine , and b = -aminoisobutyric 
 acid 
 ( aib ) . the fmoc - protected amino acids and the rink 
 amide mbha resin ( 4-(2,4-dimethoxyphenyl - fmoc - aminomethyl)-phenoxy - acetamido 
 mbha , grain size : 100200 mesh , substitution 0.83 mmol / g ) were 
 obtained from chem - impex international . sodium dodecyl - d25 used in nmr experiments was purchased from cdn isotopes 
 inc . , canada . all other reagents and solvents were purchased from 
 aldrich co. and used without further purification . fmoc construction of the glycopeptides 
 using dic / hobt coupling with microwave heating was applied to mbha - functionalized 
 rink polystyrene resin . treatment with h2nnh2h2o was required to remove the acetates from the 
 glycoside moiety prior to cleavage from the rink resin using tfa / phoch3/et3si / h2o in ch2cl2 to provide the c - terminal amides . the glycopeptides were synthesized manually using established solid - phase 
 fmoc - chemistry methodology with rink amide mbha resin ( substitution : 
 0.83 meq / g , 1% dvb ) . the side - chain - protected amino 
 acids used in the synthesis were fmoc - lys(boc)-oh , fmoc - glu(otbu)-oh , 
 fmoc - asn(trt)-oh , fmoc - d - thr(but)-oh , and fmoc - tyr(but)-oh . coupling of all the fmoc - protected amino acids was performed in a 
 sealed tube heated by an emerson 900 w microwave oven at power level 
 1 for 10 consecutive minutes . coupling was performed ( 2.0 equiv fmoc - aa 
 compared to resin ) using 1-hydroxybenzotriazole ( hobt , 2.0 equiv ) 
 and n , n-diisopropylcarbodiimide 
 ( dic , 2.0 equiv ) in a 1:1 mixture of dimethylformamide ( dmf ) and n - methylpyrrolidone ( nmp ) ( scheme 1 ) . the 
 fmoc groups were removed using a mixture of 3% piperidine and 2% diaza-1,3-bicyclo[5.4.0]-undecane 
 ( dbu ) in dmf for 10 min with argon bubbling as agitation . the final 
 fmoc deprotection as well as the acetyl protecting groups of sugar 
 moiety were removed by 80% hydrazine hydrate ( h2nnh2h2o ) in ch3oh with argon agitation 
 3 for 2 h. the glycopeptides were cleaved from the resin with 
 a f3ccooh / et3sih / h2o / phoch3/ch2cl2 ( 8:0.5:0.5:0.05:1 ) cocktail , which 
 simultaneously removed the side chain protecting groups . the crude 
 glycopeptides were precipitated in cold et2o , redissolved 
 in a minimal amount of distilled h2o , and then lyophilized . the crude glycopeptides were purified by rp - hplc on a preparative 
 c-18 phenomenex ( 250  21.9 mm ) column using ch3cn homogeneity 
 of the pure glycopeptides ( 95% ) was confirmed by analytical 
 rp - hplc and high - resolution mass spectrometry . to determine the affinity 
 and selectivity of the peptides for the - , - , and -opioid 
 receptors , chinese hamster ovary ( cho ) cells that stably expressed 
 one type of human opioid receptor were used as previously described . cell membranes were incubated at 25 c 
 with the radiolabeled ligands in a final volume of 1 ml of 50 mm tris - hcl , 
 ph 7.5 . incubation times of 60 min were used for the -selective 
 peptide [ h]damgo and the -selective ligand [ h]u69,593 , and a 3 h incubation was used with the -selective 
 antagonist [ h]naltrindole . the final concentrations of 
 [ h]damgo , [ h]naltrindole , and [ h]u69,593 were 0.25 , 0.2 , and 1 nm , respectively . nonspecific binding 
 was measured by inclusion of 10 m naloxone for the - 
 and -opioid receptors and 100 m naloxone for the -opioid 
 receptors . 32 glass - fiber filters using a brandel 
 48-well cell harvester . the filters were washed 3 with 3 ml 
 of cold 50 mm tris - hcl , ph7.5 , and were counted in 2 ml of scintisafe 
 30% scintillation fluid ( fisher scientific , fair lawn , nj ) . for [ h]u69,593 binding , the filters were soaked in 0.1% polyethylenimine 
 for at least 30 min before use . each experiment was performed in triplicate 
 and included 12 different concentrations of the competing compound . ic50 values were 
 calculated by least - squares fit to a logarithm - probit analysis . ki values of unlabeled compounds were calculated 
 from the equation ki = ( ic50)/1 + s , where s = ( concentration of radioligand)(kd of radioligand ) . all circular dichroism ( cd ) spectra 
 were obtained on olis dsm-20 automatic recording spectrophotometer 
 equipped with temperature controller . the glycopeptide stock solutions 
 were prepared by weighing the lyophilized powder using a cahn / ventron 
 instruments model 21 automatic analytical electrobalance . all cd 
 spectra were the average of three scans recorded with baseline correction 
 between 190 and 250 nm using an integration time of three seconds 
 and a scan step of 0.5 nm in a cell with a path length of 0.1 cm at 
 20 c . the molar ellipticities were calculated using the 
 equation [  ] = [ ]obs(mrw)/10lc , where [ ]obs is the observed ellipticity in millidegrees , 
 mrw is the mean residue weight , l is the cell path 
 length in centimeters , and c is the glycopeptide 
 concentration in milligrams per milliliter . the percent -helicity 
 was determined using the equation % helix = [ ]n  p*/40  000(1 
  2.5/n)100 , where n represents 
 the number of amide bonds ( including the c - terminal amide ) in the 
 glycopeptides and [ ]n  p * is the molar ellipticity 
 of the n the concentration of glycopeptide 
 samples for the nmr experiments varied from 2.5 to 3 mm . the micelle 
 samples were prepared by dissolving the peptide and 50 equiv of perdeuterated 
 sds in 0.5 ml of phosphate buffer ( 10 mm)/d2o ( 9:1 ratio 
 by volume ) . the acidity of the each sample was adjusted to ph 5.5 
 using naoh as necessary . internal standard 3-(trimethylsilyl)-d4-propionic acid ( tsp ) rotating - frame overhauser enhancement ( roesy ) , nuclear overhauser enhancement ( noesy ) , and 
 total correlation spectra ( tocsy ) were 
 acquired using standard pulse sequences and processed using xwinnmr 
 ( bruker inc . ) and felix2000 ( accelrys inc . , san diego , ca ) . mixing 
 times were 100 ms for tocsy spectra and 300 ms for roesy and noesy 
 spectra . all nmr experiments were 750 increments in t1 , 24/32/32 scans 
 each , and 1.5 s relaxation delay . molecular 
 distance constraints 
 for the structure calculation were obtained from integral volumes 
 of the roesy or noesy peaks with using software felix2000 , and the 
 noe integral volumes were classified into strong , medium , and weak 
 with 1.0 , 2.5 , and 3.5  as upper - bound distance . molecular dynamics 
 simulation was performed with the moe software ( molecular operating 
 environment , chemical computing group , canada ) using a standard protocol 
 available within the system . distance 
 constraints are placed between protons identified through nmr - determined 
 noe - corresponding upper - boundary distances of 3 ( strong ) , 4 ( medium ) , 
 and 5  ( weak ) . a 25 kcal / mol energy penalty was used for the 
 constraints . the structure was minimized initially using steepest 
 descent followed by the conjugate gradient algorithm . adult 
 male cd-1 mice ( 2535 g ) were obtained from charles river laboratories 
 and housed in groups of 4 to 5 animals / cage . animals were kept on 
 a 12 h light  dark cycle ( lights on 0700 h ) with food and water 
 available ad libitum until the time of formal testing / drug administration . they were maintained under standard housing conditions ( temperature 
 22  2 c and relative humidity between 55 and 60% ) . all 
 experimental procedures were approved by the university of new england 
 institutional animal care and use committee ( iacuc ) and were conducted 
 in compliance with the nih guide for the care and use of laboratory 
 animals . the warm - water tail - flick assay was used to assess potency 
 and efficacy of the test compounds . the assay used is a modified version of the classic tail - flick test developed by 
 damour and smith . mice were lightly 
 but firmly grasped by the nape of the neck with the evaluators thumb 
 and fingers , and the distal half of the tail was then dipped into 
 a bath of circulating water thermostatically controlled at 55 c 
 ( neslab circulator ) . latency to respond to the heat stimulus with 
 a vigorous flexion of the tail was measured to the nearest 0.1 s. 
 a baseline determination was made followed by testing at various times 
 after drug injection ( 10 , 20 , 30 , 45 , 60 , 90 , and 120 min ) . antinociception 
 was calculated by the following formula : % antinociception = [ ( test 
 latency  baseline latency)/(10 for graphing purposes , mean and sem values were calculated in excel 
 for each treatment group and time point . the a50 values 
 and 95% confidence intervals ( ci ) were calculated using linear regression 
 software from the dose  response curves ( flashcalc software ; 
 dr . michael ossipov , university of arizona , tucson , az ) . because latencies 
 in this test are affected by tail skin temperature , careful attention was paid to ensure that the ambient room 
 temperature was maintained at 22 to 23 c . briefly , mice were lightly anesthetized with 
 ether , and a 5 mm incision was made along the midline of the scalp . an injection was made using a 25 l hamilton syringe at a point 
 2 mm caudal and 2 mm lateral from bregma . all systemic injections 
 were given in a volume based on the weight of the animal ( 0.1 ml/10 
 g bodyweight ) . for intravenous ( i.v . ) injections , mice were restrained 
 in a plexiglas holder , and the distal portion of the tail was dipped 
 into 40 c warm water for approximately 10 s to dilate the tail 
 vein . the injection was made into the tail vein using a 30 gauge needle 
 and a 1 ml syringe . because 
 circular dichroism ( cd ) spectra reflect the peptide ensemble average 
 of the alignment of the dipoles of the helix backbone , this simple 
 but powerful technique can be used to obtain the secondary structures 
 in both peptides and proteins quantitatively . the cd spectra provide overall conformation but do not yield residue - specific 
 information . the helicity of the peptide 
 sequences were measured in the three different solvent systems : h2o buffer , h2o / cf3ch2oh , and 
 h2o / sds micelles . see the supporting 
 information for details of the cd data collection and interpretation . in distilled h2o buffered to ph 5.5 , the peptides and 
 glycopeptides were largely unstructured . in the presence of sds , ( g1g7 ) and mainly depended on the amino acids present 
 in the address segment ( figure 2 ) . even higher 
 helicities were observed for the unglycosylated peptides ( u1u7 ) , the most helical of which ( u1 , 70% ) was not soluble in water in the absence of sds . the lactosides 
 ( l1l7 ) were less helical than the 
 glucosides ( g1g7 ) . the highest helicities 
 were observed with sequences bearing two helicogenic -aminoisobutyric 
 acid ( aib ) residues ( u1 , g1 , and l1 ) and the lowest , sequences bearing two glycine residues in the address 
 segment ( u7 , g7 and l7 ) , with 
 the alanine - bearing sequences showing intermediate helicities . in 
 each case , increasing the degree of glycosylation ( ser[oh ]  
 ser[-glc ]  ser[-lact ] ) reduced the degree of 
 helicity . however , the identity of the sugar moieties ( glucoside vs 
 lactoside ) did not significantly affect the conformation of the micelle - bound 
 glycopeptides , causing only slight changes in the observed noe s 
 ( see nmr results later ) . the cd spectra in the 30% tfe / h2o solvent mixture showed very similar trend as the micelle - bound 
 compounds but with somewhat reduced helicities . ( a ) 
 only small degrees of helicity ( 12% max ) were observed in h2o buffer ( ph 5.5 ) and only with the unglycosylated peptides , u. ( b ) 
 in the presence of micelles , increased methylation at positions 9 
 and 12 in the address sequence ( gly  ala  aib ) led 
 to increased helicity ( 70% max ) . increased glycosylation ( -oh  
 glucose  lactose ) led to reduced helicity . circular 
 dichroism reflects 
 general information on the overall molecular structure of glycopeptides 
 in different solvents . in contrast , nmr spectroscopy is well - suited 
 to study the local structure at a residue - specific level . all of the 
 peptides and glycopeptides were characterized for their conformation 
 in aqueous buffer and in deuterated sds micelles ( peptide / sds  
 1:100 ) using 2d h nmr ( 600 mhz ) . the spin systems were 
 identified with tocsy , and sequential assignments were made by the 
 combined use of tocsy and noesy for experiments done in d25sds / d2o / h2o and roesy for experiments 
 done in d2o / h2o . although a few overlapping 
 peaks were observed , unambiguous h chemical shift assignments 
 of all glycopeptides were completed on the basis of the sequential 
 noe measurements that were made , for example , dnn ( i , i + 1 ) , dn ( i , i + 1 ) , and dn ( i , i + 1 ) . the complete chemical shift values of the amino acid residues and 
 coupling constants for all glycopeptides are provided in the supporting information along with a complete 
 description of the nmr experiments . proton chemical shift indices 
 ( csi ) for the  positions were consistent with the helix assignments 
 made by noe data . the observed chemical 
 shift differences between the ideal helix and random coil csi values 
 expected and the observed csi values were consistent with the cd data 
 for each molecule . almost all of the ch resonances showed negative 
 deviations except for residues leu and 
 ser . this trend was observed for all 
 the glycopeptides ; however , it should be noted that there is no random - coil 
 reference standard available for glycosylated serine . however , it 
 seems reasonable to make a qualitative comparison of helix content 
 between closely related peptides . first , the 
 average conformational shift was 
 calculated for each peptide by adding all upfield shifts in the helical 
 regions and dividing by the total number of peptide bonds . then , to 
 obtain the overall helical contents for each peptide , the average 
 conformational shift was divided by 0.35 ppm , which was assigned for 
 100% helicity . because there are no random - coil values available for 
 the serine glycosides and -amino 
 isobutyric acid has no -protons , these residues all of the peptide backbones exhibited strong 
 consecutive dn ( i , i + 1 ) noes 
 in h2o / d2o and in the presence of sds micelles . unlike what was observed in h2o / d2o , a continuous 
 stretch of sequential , strong dnn ( i , i + 1 ) noes were observed throughout all sequences in the 
 presence of sds micelles . in h2o / d2o , the dnn ( i , i + 1 ) noes were too 
 weak to be observed , and no other long - range noes were observable , 
 suggesting that random - coil conformational ensembles exist in this 
 solvent , as suggested by the cd data , or only nascent helix formation 
 at best . cd reflects an instantaneous snapshot of the entire ensemble , 
 whereas the nuclear overhauser effects ( noes ) take 50100 s 
 to build , depending on the peptide backbone to hold a particular conformation 
 for a relatively long period of time . thus , the helical content obtained 
 by nmr for a typical case , glycopeptide g1 ( -glucoside ) , 
 was less than 20% of cd results . because the noes were obtained by 
 true noesy experiments ( not roesy ) , the nmr structures are biased 
 toward the more static , micelle - bound structures at the expense of 
 the more dynamic , random - coil structures found in solution . thus , 
 the nmr data is useful insofar as it confirms the existence of helices , 
 but it is not useful for quantifying the degree of helicity , which 
 is more reliably predicted by the cd experiments . the peptides ( u1u7 ) and glycopeptides ( l1l7 and g1g7 ) all showed 
 low nanomolar affinities ( figure 3 and table 2 ) for the three classical opioid receptor subtypes , 
  ( hmor ) ,  ( hdor ) , and  ( hkor ) , using published 
 methods . binding was determined in membranes 
 from chinese hamster ovary ( cho ) cells that stably expressed either 
 the human - , - , or -opioid receptors . each membrane 
 preparation was incubated with 12 different concentrations of each 
 peptide / glycopeptide . each measurement was performed in triplicate , 
 and each experiment was replicated three times . figures 4 and 5 include 
 the a50 values of 
 each peptide in the 55 c tail - flick test following i.c.v . or 
 i.v . administration . the lactosylated peptides tended to be more potent 
 than both the glucosylated and unglycosylated compounds , with a50 scores ranging between 0.1 and 0.9 nmol / mouse ( figure 4 ) . administration in the 55 c 
 tail - flick assay at the doses tested ( 10 or 32 mg / kg , figure 5 ) . in contrast , most of the glycosylated analogues 
 produced potent full - agonist effects , with a50 values for 
 the disaccharides ranging from < 1 to 5.3 mol / kg . the one 
 exception was l7 ( lactosylated gg ) , which produced < 40% 
 antinociception at 32 mg / kg . higher doses of l7 were 
 not tested because there were insufficient amounts of compound available . administration . 
 mouse tail - flick 
 studies were performed at 55 c . the vertical axis ( a50 values ) the a50 values 
 were calculated using linear regression software ( flashcalc ) , and 
 95% confidence intervals are included in the table . the a50 values were calculated using linear regression 
 software ( flashcalc ) , and 95% confidence intervals are included in 
 the table . no significant antinociception was observed 
 for the unglycosylated peptides nor for the least helical lactoside 
 ( gg ) . it 
 was hypothesized that modulation of membrane affinity ( via alterations 
 in the degree of amphipathicity ) is important for bbb penetration 
 rates , for drug distribution properties , and for receptor affinity . the amphipathic character of the helix can facilitate a 
 drug or hormone to bind its specific receptor by narrowing the receptor 
 search from an inefficient 3d search of the extracellular milieu to 
 a much more rapidly converging 2d search along the membrane surface . second , membrane insertion of the helical address might allow the 
 pharmacophore or message to be fixed in a specific geometry relative 
 to the membrane . it is known that simply 
 producing highly amphipathic sequences is insufficient to facilitate 
 systemic delivery and penetration of the bbb . the -methylation of amino acids is well - known 
 to stabilize 
 helix formation and was used to produce a series 
 of helical address regions with 
 increasing intrinsic stability by increasing methylation ( glycine 
  alanine  -aminoisobutyric acid ) . in all three 
 series of opioid agonists , u1 - 7 , g1 - 7 , and l1 - 7 ( unglycosylated , 
 glucosylated , and lactosylated , respectively ) , the cd and nmr data 
 showed increasing helicity in the presence of tfe or sds . increasing glycosylation ( no sugar  glycoside  lactoside ) decreased 
 the observed helicity in every case . we hypothesize that this occurs 
 not because the helix is destabilized but by further stabilizing the 
 random - coil structures by increasing their water solubility . although 
 we can gauge the overall ratio of bound versus unbound helices , we 
 do not know the on and off rates for the helix  micelle binding 
 event ( a surrogate for helix  membrane binding ) . it would be 
 surprising if these rates were not affected by the glycosylation state . we hypothesize that the glycopeptides gain entry to the cns by transcytosis 
 at the bbb and that a minimal level of membrane interaction ( residence 
 time ? ) is required for efficient bbb penetration . desorption 
 might lead to enhanced bbb penetration rates , either by affecting 
 the biophysics of the initial endocytotic event or by promoting subsequent 
 endosomal escape of the glycopeptides . on the basis of the receptor 
 binding results and intracerebroventricular 
 ( i.c.v . ) ( figure 4 ) and intravenous ( i.v . ) 
 tail - flick studies ( figure 5 ) , only the glucoside g3 ( ba ) displayed somewhat greater receptor binding affinity 
 and in vivo potency compared to the compounds that had similar bioactivities . a50 value calculations , the compound 
 presumably exhibited efficient penetration across the bbb in mice . all lactosides ( l series ) had higher binding affinities with - , 
 - , and -receptors than the corresponding glucosides 
 ( g series ) after i.c.v injection . nevertheless , the more flexible 
 lactoside l7 ( gg ) could not , apparently , cross the bbb 
 at all after i.v . results , the lactosides were 210 times 
 more potent than the glucosides , presumably because of increased water 
 solubility within the cns . compared to tail flick results after i.v . injection , the more hydrophilic peptide l2 was 3 
 times more potent than peptide g2 , but l3 showed similar potency to g3 after peripheral administration . lactoside l7 with random - coil conformations in tfe and 
 sds micelles likely does not penetrate the bbb after i.v . injection , 
 even though the compound has a greater antinociceptive potency than g7 after i.c.v . injection . presumably , it is too water - soluble 
 and does not bind to membranes strongly enough to undergo endocytosis 
 to penetrate the bbb . none of the unglycosylated peptides showed antinociceptive 
 properties when administered peripherally ( figure 5 ) . all of the glycopeptides and peptides had 
 relatively high affinity 
 for the three cloned opioid receptors , ( table 2 ) and displayed good in vivo antinociception following i.c.v . this supports earlier conclusions that glycosylation 
 of smaller enkephalin - based peptides increases bioactivities and penetration of the bbb . both cd and nmr studies confirmed that all the glycopeptides displayed 
 random - coil conformational ensembles in aqueous solution and increasing 
 degrees of helicity in tfe and sds solution predicted by increasing 
 substitution of residues 9 and 12 , gly  ala  aib . if there were no helix - destabilizing glycine 
 residues on the address segment , then glycopeptides showed clear amphipathic 
 -helical structures by cd and by nmr . none of the unglycosylated 
 peptides showed antinociceptive properties when administered peripherally 
 ( figure 5 ) . the helix of peptide u1 was so stable that it was not even soluble in aqueous media in the 
 absence of sds . the results suggest that simply introducing 
 highly helical sequences 
 on the address segment is not by itself sufficient to promote stability 
 and penetration of the bbb . it is hypothesized that the helix must 
 also be capable of assuming a water - soluble random - coil conformation 
 and that the energy barrier between random coil and helical states 
 must be low enough to permit rapid interconversion between the two 
 states ; this characteristic was termed biousian , denoting two ( bi ) 
 essences ( ousia ) , a water - soluble conformational ensemble and a membrane - bound 
 conformation . the biousian nature of 
 a glycopeptide permits high - affinity receptor binding , allows membrane 
 hopping to impart drug - like characteristics , and promotes penetration 
 of the bbb . this is confirmed by comparing glucoside g2 and lactoside l2 ( table 2 ) . the cd spectra of g2 indicates a strong helix but a relatively 
 low affinity for opioid receptors and a weak penetration of the bbb . the increased hydrophilicity of the lactose - bearing l2 ( decreased energy barrier between random coil and helical states ) 
 allows l2 to bind more strongly than g2 and 
 to penetrate the bbb after i.v . unglycosylated peptide u1 showed excellent opioid binding , yet it was less antinociceptive 
 than u2 or u3 after i.c.v . administration 
 ( figure 4 ) and was not water - soluble in the 
 absence of sds . further studies with other opioid messages ( e.g. , 
 - and -selective agonists ) and replacement of the linkage 
 element l - proline with more flexible linkers will be discussed 
 elsewhere .
glycosylated 
 -endorphin 
 analogues of various amphipathicity were studied in vitro and in vivo 
 in mice . 
 opioid binding affinities of the o - linked glycopeptides ( mono- 
 or disaccharides ) and unglycosylated peptide controls were measured 
 in human receptors expressed in cho cells . 
 all were pan - agonists , 
 binding to - , - , or -opioid receptors in the 
 low nanomolar range ( 2.235 nm ki s ) . 
 the glycoside moiety was required for intravenous ( i.v . ) 
 but not for intracerebroventricular ( i.c.v . ) 
 activity . 
 circular dichroism 
 and nmr indicated the degree of helicity in h2o , aqueous 
 trifluoroethanol , or micelles . 
 glycosylation was essential for activity 
 after i.v . 
 administration . 
 it was possible to manipulate the degree 
 of helicity by the alteration of only two amino acid residues in the 
 helical address region of the -endorphin analogues 
 without destroying - , - , or -agonism , but the 
 antinociceptive activity after i.v . 
 administration could not be directly 
 correlated to the degree of helicity in micelles .