Patent Application: US-78784099-A

Abstract:
this invention relates to methods for preparing cyclic peptides and peptidomimetic compounds in solution and bound to solid supports , and to cyclic peptide or peptidomimetic libraries for use in drug screening programmes . in particular , the invention relates to a generic strategy for synthesis of cyclic peptides or peptidomimetics that enables the efficient synthesis under mild conditions of a wide variety of desired compounds . two approaches were evaluated for their improvements in solution and solid phase synthesis of small cyclic peptides : positioning reversible n - amide substituents in the sequence ; and applying native ligation chemistry in an intramolecular sense . systematic investigation of the effects of preorganizing peptides prior to cyclisation by using peptide cyclisation auxiliaries , and developing new linkers and peptide cyclisation auxiliaries to aid cyclic peptide synthesis gives surprising improvements in both yields and purity of products compared to the prior art methods . the combination of these technologies provides a powerful generic approach for the solution and solid phase synthesis of small cyclic peptides . the ring contraction and n - amide substitution technology of the invention provide improved methods for the synthesis of cyclic peptides and peptidomimetics . when used in conjunction with linker strategies , this combination provides solid - phase avenues to cyclic peptides and peptidomimetics .

Description:
the invention will now be described in detail by way of reference only to the following non - limiting examples . acoh acetic acid boc n α - tert - butoxycarbonyl bop benzotriazole - 1 - yl - oxy - tris -( dimethylamino )- phosphonium - hexafluorophosphate t bu tert - butyl calcd calculated dcm dichloromethane diea diisopropylethylarnine dmf n , n - dimethylformamide eq equivalent es - ms electrospray mass spectrometry lc / ms liquid chromatoraphy linked to mass spectrometry fmoc 9 - fluorenylmethyloxycabonyl - hbtu o - benzotriazole - n , n , n ′, n ′- tetramethyluronium hexafluorophosphate hf anhydrous hydrogen fluoride nmr nuclear magnetic resonance onbs o - nitrobenzene - sulfonamide pam phenylacetamidomethyl pmc 2 , 2 , 5 , 7 , 8 - pentamethylchroman - 6 - sulfonyl rp - hplc reversed phase high - performance liquid chromatography rt room temperature spps solid phase peptide synthesis tfa trifluoroacetic acid uv ultraviolet chlorotrityl resin ( sv = 0 . 92 mmol / gr ) was purchased from pepchem ( tubingen , germany ). all wang resins and n α - tert - butoxycarbonyl ( boc )- l - amino acids were peptide synthesis grade purchased from auspep ( melbourne australia ), novabiochem ( san diego ) or peptide institute ( osaka , japan ). pam resins were purchased from applied biosystems ( foster city , calif .). dichloromethane ( dcm ), diisopropyl - ethylamine ( diea ), n , n - dimethylformamide ( dmf ), and trifluoroacetic acid ( tfa ) were obtained from auspep ( melbourne , australia ). p - cresol , p - thiocresol , 3 - nitrophenol , polyphosphoric acid , hexamethylenetetramine were purchased from aldrich or fluka ( sydney , australia ). hplc grade acetonitrile was purchased from millipore - waters ( sydney , australia ). 2 -( 1h - benzotriazol - 1 - yl )- 1 , 1 , 3 , 3 - tetramethyluronium hexafluoro - phosphate and benzo - triazole - 1 - yl - oxy - tris -( dimethylamino )- phosphonium - hexafluoro - phosphate was purchased from richelieu biotechnologies ( quebec , canada ). deionised water was used throughout and was prepared by a milli - q water purification system ( millipore - waters ). screw - cap glass peptide synthesis reaction vessels ( 20 ml ) with sintered glass filter frit were obtained from embell scientific glassware ( queensland , australia ). argon , helium and nitrogen ( all ultrapure grade ) were from boc gases ( queensland , australia ). 1 h nmr and 13 c nmr spectra were recorded on a varian 300 mhz gemini in cd 3 od , and chemical shifts are reported in parts per million ( ppm ) downfield from ( ch 3 ) 4 si . reversed phase high - performance liquid chromatography ( rp - hplc ) was performed on a waters 600e solvent delivery system equipped with a 484 absorbance detector at 214 nm or 230 nm ( applied biosystems inc .) or on a hewlet packard hp1100 system equipped with diode - array detector . hplc data were collected using turbochrome or hplc 2d chemstation software . rp - hplc was performed on a zorbax microbore ( sb - c18 , 2 . 1 mm × 5 cm ) column , a vydac c - 18 analytical ( 5 mm , 0 . 46 cm × 25 cm ) column or a vydac preparative c - 18 ( 10 mm , 2 . 2 cm × 25 cm ) column . chromatographic separations were achieved using linear gradients of buffer b in a ( a = 0 . 1 % aqueous tfa ; b = 90 % ch 3 cn , 10 % h 2 o , 0 . 09 % tfa ) at a flow rate of 0 . 25 ml / min ( microbore ), 1 ml / min ( analytical ) and 8 ml / min ( preparative ). mass spectra were acquired on a pe - sciex api - iii triple quadrupole mass spectrometer equipped with an ionspray atmospheric pressure ionization source . samples ( 10 μl ) were injected into a moving solvent ( 30 μl / min ; 50 / 50 ch 3 cn / 0 . 05 % tfa ) coupled directly to the ionisation source via a fused silica capillary interface ( 50 mm i . d .× 50 cm length ). sample droplets were ionized at a positive potential of 5 kv and entered the analyser through an interface plate and subsequently through an orifice ( 100 - 120 mm diameter ) at a potential of 80 v . full scan mass spectra were acquired over the mass range of 400 to 2000 daltons with a scan step size of 0 . 1 da . molecular masses were derived from the observed m / z values using the macspec 3 . 3 and biomultiview 1 . 2 software packages ( pe - sciex toronto , canada ). calculated theoretical monoisotopic and average masses were determined using the macbiospec program ( pe - sciex toronto , canada ). lc / ms runs were carried out using a linear gradient on a 140b abi dual syringe pump solvent delivery system and a zorbax reversed phase c - 18 ( sb , 2 . 1 mm × 5 cm )) column at a flow rate of 150 μl / min . samples ( typically 5 μl of 1 mg / ml solution ) were loaded directly on the column and the eluent directly connected to the mass spectrometer via a 30 cm , 75 mm i . d . fused silica capillary . the application of turbo ionspray ™ ( 5 l / min n2 at 500 ° c .) allowed the introduction of the total eluent without splitting and loss in sensitivity . acquisition parameters were as described above . our synthetic approach for introducing the auxiliary is depicted in scheme 9 . in brief , there are two reported pathways that provide access to the n - substituted linear peptides : 1 . a first , and most common route , makes use of a reductive alkylation of the n - terminal primary amine with the aldehyde . initially the aldehyde is added in excess to the resin - bound peptide . after imine formation is complete , as assessed by ninhydrin reaction , a solution of nabh 4 in dmf / meoh ( 2 / 1 ) or nabh 3 cn in dmf / meoh ( 1 / 1 ) containing 5 % acetic acid is added to reduce the imine and generate the — ch 2 — link between the auxiliary and the amine . 2 . in a second route an o - nitrobenzene - sulfonamide ( onbs ) is prepared from the corresponding sulfonylchloride and the amine . a mitsonobu - type alkylation , using the auxiliary alcohol , establishes the link with the amine . finally the onbs - group is removed using phsna in dmf . this three - step process is also known as the fukuyama reaction . synthesis was performed on fmoc - gly - wang resin ( 0 . 36 mmol / g ). the tetrapeptide tyr - arg - phe - gly ( seq id no : 21 ) was assembled using stepwise fmoc - spps , with alternating hbtu coupling and piperidine deprotection as follows : 2 mmol fmoc - amino acid ( 4 equivalents ) were dissolved in dmf ( 4 ml ) and 2 mmol hbtu added to the solution together with diea ( 400 μl ). after 1 minute preactivation the solution was added to the amino - resin . the reaction was left for 10 minutes and ninhydrin test performed on a small resin sample to measure the amount of remaining free amine . if the measured amount of free amine was more than 0 . 5 %, the reaction was left longer ( 30 minutes ) and if necessary repeated . the resin was then washed several times with dmf . the fmoc protected resin ( 0 . 5 mmol ) was treated with a solution of piperidine / dmf ( 1 / 1 ) ( 4 ml ) for 2 minutes . the resin was drained and the piperidine treatment repeated . the resin was then washed several times with dmf before the next coupling step was commenced . the tyr ( bu )- arg ( pmc )- phe - gly - wang resin ( seq id no : 2 ) ( 1 g ) was then treated with s -( p - methylbenzyl )- 2 - mercapto - acetaldehyde ( 58 mg ; 0 . 32 mmol , bitan et al , 1997 ) dissolved in meoh / dmf / acoh ( 47 / 47 / 5 ) ( 6 ml ). after 5 min stirring 60 mg of nabh 3 cn was added and the mixture left for 60 minutes . the reductive alkylation step was then repeated once more to ensure complete reaction . the resin was washed several times with dmf / meoh , meoh / dcm and dcm and finally air dried . 1 g of resin was treated with 1 ml of p - cresol and 9 ml hf at 0 ° c . for 1 hour . the hf was evaporated in vacuo and the residue triturated with cold diethylether ( 20 ml ). the ether was filtered off and the precipitated dissolved in a small amount of water . the solution was then loaded directly on to an hplc column ( vydac , c18 reverse phase 2 . 1 × 25 cm ) for hplc purification of the products ( buffer a : water , 0 . 1 % tfa ; buffer b : acetonitrile / water 1 / 1 , 0 . 09 % tfa ) ( 100 % a to 80 % b in 60 min ). the hs —( ch 2 ) 2 — nh — ch ( ch 2 phoh )— co - arg - phe - gly - oh was isolated and lyophilised , yielding 70 mg of white powder ( 45 % yield ), mr 601 . 5 , calcd for c 28 h 39 n 7 o 6 s : 601 . 27 . tyr ( bu )- arg ( pmc )- phe - gly - wang resin ( seq id no : 2 ) was prepared as described in example 1 . 120 mg of 2 , 2 ′- dialdehyde - 4 , 4 ′- dinitro -[ diphenyldisulfide ] ( fries and brothuhn , 1923 ) ( 0 . 33 mmol ) was dissolved in meoh / dmf / acoh ( 47 / 47 / 5 ) ( 3 ml ) and the solution added to the resin . after 5 minutes , 100 mg nabh 3 cn ( 1 . 6 mmol ) was added and the solution stirred for 15 hours . the resin was washed with dmf ( 3 ×) and meoh / dcm 1 / 1 ( 3 ×). 700 mg of resin was treated with 10 ml tfa / h 2 o ( 95 / 5 ) for 1 hour at room temperature . the tfa was removed in vacuo and the residue dissolved in hplc buffers a / b 1 / 1 ( 5 ml ). the solution was then loaded directly on to an hplc column and purification of the product performed as in example 1 . 25 mg n -( 5 - nitro - 2 - mercaptobenzyl )- tyr - arg - phe - gly - oh ( seq id no : 3 ) were obtained from lyophilisation ( 20 % yield ), mr 708 . 4 ( calcd for c 33 h 40 n 8 o 8 s : 708 . 27 ). ala - phe - leu - pro - ala - wang resin ( seq id no : 5 ) was prepared starting from fmoc - ala - wang resin ( 0 . 44 mmol / gram ) using standard fmoc - spps protocols , with hbtu coupling and piperidine deprotection as described in example 1 . to 500 mg of this resin , a solution of 300 mg o - nitrobenzenesulfonylchloride in dmf ( 4 ml ) containing diea ( 200 μl ) was added . after 30 minutes , the resin was drained and washed with dmf ( 3 ×). the resin was mixed with a solution of s -( p - methylbenzyl )- 2 - mercaptoethanol ( 270 mg , 1 . 5 mmol ) in dcm ( 5 ml ). triphenylphosphine ( 393 mg , 1 . 5 mmol ) and diethylazodicarboxylate ( dead , 261 mg , 1 . 5 mmol ) were premixed in dcm ( 5 ml ). after 1 minute , the solution was added to the resin and the reaction left for 30 minutes . the resin was washed with dcm ( 3 ×) and dmf ( 3 ×). the resin was further treated with a solution of nasphe ( 200 mg , 1 . 5 mmol ) in dmf ( 4 ml ) for 30 minutes . the resin was washed with dmf ( 3 ×) and meoh / dcm ( 3 ×) and air dried . 500 mg of resin was cleaved using hf / p - cresol / p - thiocresol ( 9 / 1 / 1 ) ( 10 ml ) ( 1 hour at 0 ° c .) and was worked up as described in example 1 . the crude residue was dissolved in buffers a / b ( 1 / 1 ) and purified on hplc yielding hs —( ch 2 ) 2 — nh — ch ( ch 3 )— co - phe - leu - pro - ala - oh ( seq id no : 6 ) ( 25 mg , 22 % yield ). mr : 577 . 1 ( calc for c 28 h 43 n 5 o 6 s : 577 . 29 ) fmoc - ala - trityl resin ( 0 . 4 mmol / gr ) was first prepared from trityl resin ( 0 . 96 mmol / gr ) using protocols provided by pepchem ( tubingen , germany ). ala - phe - leu - pro - ala - trityl resin ( seq id no : 36 ) was assembled using standard fmoc spps protocols , as in example 1 . this resin ( 0 . 5 gr ) was further treated with a solution of 2 - hydroxy - 5 - nitrobenzaldehyde ( 115 mg , 0 . 7 mmol ) and acoh ( 20 μl ) in dmf ( 2 ml ). after 5 minutes the resin was drained and a second aldehyde treatment was performed . the resin was drained , and washed copiously with dmf until eluent was colourless . a solution of nabh 4 ( 150 mg , 4 mmol ) in dmf / meoh 3 / 1 ( 4 ml ) was added and the resin stirred for 10 minutes . the resin was drained , washed with dmf / meoh 1 / 1 , dcm / meoh 1 / 1 and dcm and air dried . the resin was treated with dcm ( 10 ml ) and tfa ( 100 μl ) for 1 hour . the solution was evaporated , buffer b ( 3 ml ) was added and the resin filtered off . the solution was loaded directly on to a preparative hplc column and hplc purification performed using a 2 % gradient ( from 90 % a to 10 % a in 40 minutes ). after lyophilisation n -( 2 - hydroxy - 5 - nitrobenzyl )- ala - phe - leu - pro - ala - oh ( seq id no : 7 ) ( 114 mg ) was isolated as a white powder ( 85 % yield ), mr : 668 . 2 ( calcd for c 33 h 44 n 6 o 9 : 668 . 32 ). the linear peptide was synthesised on trityl resin as described in example 4 , but employing 2 - hydroxy - 6 - nitrobenzaldehyde ( harayama et al , 1994 ). after lyophilisation n -( 2 - hydroxy - 6 - nitrobenzyl )- ala - phe - leu - pro - ala - oh ( seq id no : 8 ) ( 85 mg ) was isolated as a white powder ( 63 % yield ), mr 668 . 2 , calcd for c 33 h 44 n 6 o 9 : 668 . 32 ). the most important factor limiting the use of the prior art methods was the o - to - n or s - to - n acyl transfer step . one of the parameters which we envisaged would have a large impact on the rate of acyl transfer was the activation of the intermediate oxy - or thioester bond . with this in mind we focussed on the following auxiliary modifications : introduction of a nitro substituent on the aromatic ring of the auxiliary . nitrophenyl esters react more readily with nucleophiles when compared to phenyl esters . thio - esters versus oxy - esters . in previous work in our laboratory we found that under the same conditions both esters hydrolyse at the same rate . we have included both phenols and thiophenols in our set of n - substituents . to compare the rate of acyl transfer we carried out the reactions shown in scheme 10 . a tripeptide ( ala - gly - phe ) was assembled on wang - resin , and the selected range of aldehydes introduced via reductive amination . each n - substituted tripeptide was then subjected to acylation by mixing the resin with a solution of hbtu - activated boc - amino acid ( boc - ala - oh , boc - phe - oh or boc - val - oh ). the general reaction pathway was as follows : acylation of the n - substituted tripeptide occurs initially on the phenol oxygen ( or sulfur ) atom . the acyl group subsequently migrates to the nitrogen atom . the liberated phenol ( or thiophenol ) functionality was then acylated a second time . we included as a control the 2 - chloro - 4 - nitrobenzyl substituent . due to the absence of the alcohol we expected that acylation directly on to the secondary amine , will be much slower then for the phenol / thiophenol cases . the results are summarized in table 1 . 1 . the presence of the alcohol / thiol function at the ortho position of the aromatic ring dramatically increases the rate of acylation of the substituted amine . under the same acylation conditions , the ortho - chloro “ auxiliary ” did not undergo acylation at the secondary amine site . 2 . the presence of an alcohol / thiol function in combination with a nitro substituent on the aromatic ring ensures very rapid acylation of the secondary amine . for most of the cases studied & gt ; 70 % diacylated product was formed after 10 minutes . acylation of the 2 - hydroxy - 6 - nitro - derivative with boc - valine required 60 minutes to achieve 82 % diacylation . in other words , acylation at the nitrogen atom was & gt ; 80 % complete after 60 min . when boc - valine was used . as a comparison , in a very similar study acylation of the alphα - nitrogen atom of n -[ 2 - hydroxy - 4 - methoxybenzyl ]- alanine with fmoc - valine was still incomplete after 24 hours . in the case of acylation of the 2 - mercapto - 5 - nitro - derivative with boc - alanine , diacylation only reaches 37 % after 60 min . ; from comparison with the other results we ascribe this to hydrolysis of the highly reactive and less hindered ester bond in the diacylated product during work - up . 3 . the position of the nitro - group on the ring does not seem to play a major role , although small differences can be observed . substituting oxy - ester for thioester does not significantly alter the rate . the ala - gly - phe sequence was assembled on wang resin ( 0 . 1 mmol , novabiochem ) using standard fmoc / hbtu protocols . auxiliaries were introduced as described in examples 2 , 4 and 5 . each resin was then distributed into three portions in a separate reaction vessels , and swelled in dmf for 10 min . three equivalents of boc - ala - oh , boc - phe - oh , or boc - val - oh were coupled to ala - gly - phe - resin using 0 . 99 equivalents of hbtu with 4 equivalents of diea . samples ( 10 - 15 mg ) were removed after 1 minute , 10 minutes and 60 minutes , immediately drained , washed with 1 : 1 dcm : meoh , and dried . samples were then cleaved with 250 ml of 97 % tfa at room temperature for 1 h . the tfa was evaporated with a stream of nitrogen and the product dissolved in 50 % b ( 100 ml ). the sample was centrifuged and the supernatant collected , then analysed by rp - hplc and es - ms or lc / ms . the relative yields of unreacted starting material , monoacylated and diacylated product for each experiment were calculated by integrating the hplc signals in the corresponding chromatogram . in this example we demonstrate the improved o → n acyl transfer kinetics of our auxiliaries when compared to prior art auxiliaries such as the 2 - hydroxy - 4 - methoxybenzyl auxiliary . the n - acylation product is formed from the ligation of one residue with a resin bound peptide . this example thus also serves as an example of improved ligation using the auxiliaries of this invention . in order to establish the relative o → n acyl transfer efficiencies of the 2 - hydroxy - 4 - methoxybenzyl , the 2 - hydroxy - 5 - nitrobenzyl and the 2 - hydroxy - 6 - nitrobenzyl auxiliaries , a series of 2 -( 1h - benzotriazol - 1 - yl )- 1 , 1 , 3 , 3 - tetramethyluronium hexafluorophosphate ( hbtu )- mediated amino acid acylation experiments were carried out . initially , the ortho - oxygen atom of the benzyl auxiliary is acylated by the hbtu - activated amino acid to yield 2 . following this , the acyl group then migrates to the n α - nitrogen atom to form an amide bond 3 . the ortho - oxygen in the presence of excess acylating agent then becomes available for acylation for a second time with diacylation leading to 4 ( scheme 11 ). the resin - bound tripeptide , ala - gly - phe , was assembled using standard fmoc - spps protocols on chlorotrityl resin , and the benzaldehyde auxiliary incorporated by reductive alkylation . the peptide - resin of each n α - auxiliary substituted peptide was divided into three separate portions , and subjected to acylation with 3 equiv of 0 . 5 m hbtu - activated fmoc - ala , fmoc - phe , or fmoc - val in dmf . aliquots were removed for analysis after 1 , 10 , and 60 min . following reaction , the peptide - resins were washed with dmf , base - treated with piperidine / dmf / water , washed with dmf then dcm , dried , and cleaved with 0 . 5 % tfa in dcm for 30 min . these base treatments were employed to remove o - aryl esters present ( 2 and 4 ), prior to tfa cleavage , in order to distinguish between auxiliary ( o ) and n α - amino acylation ( scheme 11 ). the products were then identified by es - ms or lc / ms analysis and quantified by reversed phase - hplc peak integration . the results are shown in table 2 . initially , to determine the extent of direct n α - acylation using hbtu - activated amino acids , acylation experiments were carried out with a control auxiliary , n α - 2 - chloro - 5 - nitrobenzyl . as expected , no appreciable amount (& lt ; 1 %) of secondary n α - acylation had occurred after 30 min with hbtu - activated alanine , phenylalanine , or valine . next , the presence of an “ inactivated ” ortho - hydroxy group as with the hmb auxiliary ( 1a ) was examined . we found the formation n α - acylated products did occur by acyl transfer , albeit at relatively slow rates . hmb - mediated acyl transfer proceeds at a reasonable rate for alanine after 1 h ( 92 %), but the transfer efficiency diminishes sharply with increasing steric bulk on amino acid side - chains , as with phenylalanine ( 48 %) or valine ( 12 %). these observations agree well with acylation studies carried out by others , and also re - emphasises the need for n - terminal auxiliaries for spps with improved acyl transfer efficiency . in contrast to the limb auxiliary ( 1a ), the presence of an ortho - hydroxyl group in combination with the electron - withdrawing nitro substituent on the benzyl ring of an auxiliary significantly enhances acyl capture and transfer efficiency . acyl transfer ( n α - acylation ) mediated by the 2 , 5 - hnb auxiliary ( 1b ) was rapid for all three amino acids . for alanine and phenylalanine , n α - acylation products formed rapidly , and the reaction was near completion after 10 min . in a similar manner to the 2 , 5 - hnb auxiliary , high alanine , phenylalanine , and valine acyl transfer efficiencies were also observed with the hnb auxiliary ( 1c ) after 1 h . importantly , for the more sterically demanding acylation of valine onto n α -( aux ) ala - gly - phe the yields with both the 2 , 5 - hnb (& gt ; 95 %) and hnb ( 88 %) auxiliaries were nearly eight - times higher than with the hmb auxiliary ( 12 %) under identical experimental conditions . the position of the nitro - substituent on the aromatic ring of the auxiliary does not appear to play a crucial role . in all cases racemisation from the acyl transfer reaction was not evident . to determine if the activating effect of the nitro substituent could also improve acyl transfer rates and yields with more sterically demanding residues a second set of acylation experiments were carried out . in these experiments , the same auxiliaries were each introduced onto the resin - bound tetrapeptide , val - ala - gly - phe ( seq id no : 9 ), by reductive alkylation and subjected to acylation by hbtu - activated fmoc - gly , fmoc - phe and fmoc - val . due to the inherent difficulty associated with the acylation of sterically hindered secondary amines the acyl transfer reaction time course in these experiments was increased to 1 , 6 , and 24 h . following acylation , the peptide - resins were subjected to piperidine base treatment to exclusively observe the n α - amino acylation products and then cleaved with 0 . 5 % tfa in dcm for 30 min . the products were identified by es - ms or lc / ms analysis and quantified by rp - hplc peak integration . the results are summarised in table 3 . distribution of n α - acylation products on the val - ala - gly - phe ( seq id no : 9 ) sequence by n α - auxiliary directed o → n acyl migration . from the acylation results listed in table 3 , it is clear that the nitro - activated auxiliaries strongly enhance o → n acyl transfer rates and yields with respect to the hmb auxiliary . in all three cases the 2 , 5 - hnb auxiliary and hnb were superior acyl transfer auxiliaries . the 2 , 5 - hnb - assisted acyl transfer of glycine onto valine proceeded rapidly , and was essentially complete (& gt ; 98 %) within 1 h , whereas phenylalanine and valine required 24 h for greater than 95 % completion . the hnb auxiliary also induced rapid acyl transfer , but nevertheless still required 24 h for approximately 95 % n α - acylation for all three amino acids . on the other hand , hmb - assisted acyl transfer resulted in unacceptably low n α - acylation yields , especially with phenylalanine and valine , even though o - acylation proceeds in approximately 50 % and 90 % yield after 1 h , respectively . more specifically , the hmb - assisted n α - acylation of valine only proceeds in 23 % yield after 24 h , and is approximately four times lower than with either the 2 , 5 - hnb ( 95 %) or hnb ( 93 %) auxiliary . these results strongly indicated that the 2 , 5 - hnb and hnb auxiliaries are of considerable value for the chain assembly of “ difficult ” peptide sequences , especially when β - branched or bulky residues are predominant . peptide synthesis . peptides were chemically synthesised stepwise , using 0 . 5 m hbtu - dmf activation protocols for fmoc and boc chemistry as previously described . the syntheses were performed on p - benzyloxybenzyl alcohol ( wang ) resin or chlorotrityl resins . the following amino acid side chain protection was used : fmoc - glu ( o - tert - butyl )- oh , fmoc - lys -( boc )- oh , fmoc - ser ( o - benzyl )- oh , fmoc - thr ( o - tert - butyl )- oh , and fmoc - tyr ( o - tert - butyl )- oh . each residue was coupled for 10 min , and coupling efficiencies determined by the quantitative ninhydrin reaction and the qualitative isatin test . reductive alkylation . typical procedure for the incorporation of the auxiliaries onto a growing peptide chain during spps . 0 . 1 mmole of the n α - deprotected peptide - resin was washed with 10 ml dmf / meoh / acoh ( 9 : 9 : 2 ), drained , and then mixed with 3 equiv of the substituted benzaldehyde in 1 ml of dmf / meoh ( 1 : 1 ) for 30 min . the solution was then drained and imine formation reaction repeated . following drainage , the resin was washed briefly with dmf , then 5 equiv of sodium borohydride in dmf / meoh / acoh ( 9 : 9 : 2 ) was added and mixed at room temperature for 5 min . the resin was then drained and washed successively with dmf , dmf / h 2 o , h 2 o , meoh / dcm , dcm , and dmf . a few milligrams (˜ 3 mg ) was then taken , dried , cleaved with 97 % aqueous tfa , dissolved in 30 % buffer b , filtered , and then immediately analyzed by es - ms and rp - hplc to determine reaction completion . acylation experiments . the ala - gly - phe and val - ala - gly - phe ( seq id no : 9 ) sequences were assembled on chlorotrityl resin ( 0 . 96 mmol / g , pepchem ) using standard fmoc / hbtu protocols . the resin was then divided into three portions in separate reaction vessels , and swollen in dmf for 10 min . three equiv of fmoc - protected glycine , alanine , phenylalanine , and valine were coupled to ala - gly - phe - resin or val - ala - gly - phe - resin ( seq id no : 37 ) using 2 . 95 equiv of 0 . 5 m hbtu in dmf with 4 equiv of diea for 1 , 10 , and 60 min or 1 , 6 , and 24 h , respectively . to examine n α - acylation exclusively and also remove fmoc groups , resins samples before cleavage were subjected to 2 cycles of 5 min piperidine / dmf ( 1 : 1 ) and 5 min dmf / piperidine / h 2 o ( 4 : 4 : 2 ) treatments , then dried with dcm : meoh ( 1 : 1 ). trityl resin samples were cleaved with 0 . 5 % tfa in dcm for 30 min . the tfa cleavage solutions were evaporated with a stream of nitrogen , and the product dissolved in 100 μl of 50 % buffer b . samples were centrifuged , supernatant collected , then immediately analysed by rp - hplc and es - ms or lc / ms . in principle , the 2 , 6 - hnb and hmb groups should have a similar effect on disrupting hydrogen - bonding networks , since they both alter the backbone structure of growing peptide chains and remove a backbone hydrogen - bond donor . to demonstrate this beneficial effect , the stat - 91 ( 699 - 709 ) sequence , tgyiktelisv ( seq id no : 38 ), which we have previously reported as “ difficult ” in both fmoc - and boc - spps ( meutermans et al 1996 ; alewood et al , 1997 ), was assembled using standard chain assembly protocols and also with the assistance of n - hnb backbone substitution under identical experimental conditions . the stat - 91 peptide was selected because it does not contain a relatively unhindered site before the “ difficult ” section is encountered , and thus precludes the use of the hmb auxiliary . using standard fmoc / tent - butyl 0 . 5 m hbtu / diea 10 min coupling protocols in dmf , the chain assembly of stat - 91 ( 699 - 709 ) proceeds well until residues glu 705 and thr 704 which only couple in 67 % and 91 % yield , respectively . this is then followed by a 59 % coupling yield at lys 703 , which reportedly only increases to 62 % upon re - coupling after 1 h . furthermore , substitution of hbtu with hatu did not significantly improve the coupling yield for this residue . tyrosine 700 also gave a poor coupling yield ( 61 %) that also does not improve significantly even after re - coupling for 1 h . in short , the chain - assembly of stat - 91 ( 699 - 709 ) under standard coupling conditions proceeded with an unacceptable average acylation yield of 83 % ( see figure below ). however , when the hnb ( 3 ) auxiliary is incorporated onto the backbone of the growing peptide at ile 707 ( the third residue from the resin - linker ) by reductive alkylation , subsequent chain - assembly proceeded in high efficiency using the same coupling conditions . with the assistance of the hnb auxiliary , the average coupling yield for the assembly of ile 707 ( n α - hnb )- stat - 91 ( 699 - 709 ) increased to 99 . 6 % as determined by the quantitative ninhydrin assay . this is illustrated in fig1 . the hbtu - mediated n α - acylation of leu 706 onto the n α - hnb - substituted ile 707 was monitored by es - ms and rp - hplc analysis of a cleaved resin sample before continuation . the fmoc - leu to n α -( hnb ) ile - ser - val - resin acyl transfer reaction was approximately 50 % complete after 1 h , but apparently quantitative after 24 h as determined by rp - hplc analysis ( data not shown ). by comparison , for the identical fmoc - leu to n α -( hmb ) ile - ser - val - resin acyl transfer reaction , n α - acylation only proceeds in 21 % yield after the same period . this poor hmb - assisted n α - acylation yield further highlights the limitation of the hmb auxiliary in the synthesis of hindered or β - branched containing “ difficult ” peptides . following chain - assemblies , the crude tfa cleavage material of the standard and hnb - assisted stat - 91 ( 699 - 709 ) syntheses were analysed by es - ms and rp - hplc . when employing standard fmoc / hbtu synthesis protocols , no appreciable amount (& lt ; 1 %) of the target stat - 91 peptide was found in the crude product by either es - ms or rp - hplc analysis . in contrast , with the hnb - assisted synthesis , the hnb - substituted stat - 91 ( 699 - 709 ) peptide was the major component in the crude cleavage material as determined by es - ms and rp - hplc . the ile 707 ( n α - hnb )- stat - 91 ( 699 - 709 ) peptide was then readily photolysed at 366 nm for 3 h to give the target peptide in good yield . from this comparison , it appears that the hnb auxiliary can be used to significantly improve the chain - assembly efficiencies of “ difficult ” peptides in a manner similar to that of hmb - substitution , but with the key advantage of greatly improved acyl transfer efficiency . stat - 91 ( 699 - 709 ) syntheses the standard and hnb - assisted synthesis of stat - 91 ( 699 - 709 ) was carried out using fmoc chemistry on fmoc - val - wang resin ( applied biosystems inc ., 0 . 70 mmol / g , 0 . 1 mmole synthesis scale ). the standard assembly using 10 min couplings times resulted in an average coupling yield of 83 %. the hnb - assisted fmoc chain - assembly of stat - 91 ( 699 - 709 ) was also performed using 10 min coupling times , with an average coupling yield of 99 . 6 %. the hnb auxiliary was introduced at ile 707 by reductive amination ( 2 × 3 h imine formation reaction using 5 equiv hnb ) and was then followed by the 0 . 5 m hbtu coupling of leu 706 in dmf . n α - acylation of leu 706 onto ile 707 ( n α - hnb )- stat - 91 ( 708 - 709 )- resin was complete after 24 h as determined by es - ms and rp - hplc analysis following tfa cleavage of small resin sample . to maintain experimental consistency , ile 707 in the standard synthesis was also coupled with hbtu for 24 h . in both cases , the final peptide - resin product was cleaved with 97 % aqueous tfa at room temperature for 1 h . after evaporation of the tfa in vacuo , the crude product was washed with cold diethyl ether ( 2 × 5 ml ), dissolved in 30 % b ( 5 ml ), and lyophilised . ile 707 ( n α - hnb )- stat - 91 ( 699 - 709 ) es - ms : m r 1374 . 1 da ; calculated for c 63 h 99 n 13 o 21 : 1373 . 7 da ( monoisotopic ); the hnb - substituted stat peptide was then photolysed as described below to give the target peptide in 76 % yield after rp - hplc purification : stat - 91 ( 699 - 709 ) es - ms : m r 1222 . 7 da ; calculated for c 56 h 94 n 12 o 18 : 1222 . 68 da ( monoisotopic ). photolysis . photolysis was carried out in a camag uv - cabinet ii , with a lamp wavelength of 366 nm and power of 0 . 25 amps for 2 - 3 h . typically 5 mg samples of peptide - resin or 1 mg samples of peptide were taken up in 500 μl , meoh in an uncovered wide - mouth vial on a white mat . h - ala - phe - leu - pro - ala - oh ( seq id no : 39 ) was a recently reported example of a sequence which is difficult to cyclise ( schmidt and langner , 1997 ). when subjected to cyclisation conditions , dimer and higher oligmers were generated , but no target cyclopentapeptide was formed . we have employed this linear peptide to probe our methodology and compare it with the prior art methods . in the following set of experiments we demonstrate that this ala - ala amide bond in the monocycle was not accessible from this linear peptide using prior art methodologies , but was accessible using our photolabile auxiliaries . as a control experiment we attempted to cyclise the unsubstituted linear peptide ( ala - phe - leu - pro - ala ) ( seq id no : 19 ) using standard cyclisation conditions ( 1 mm in dmf , 3 eq . bop , 5 eq . diea , 3 h at rt ). as expected from the previously reported results , only cyclic dimer and some trimer were obtained , but no target monocyclic product . we initially evaluated an ethanethiol auxiliary . this auxiliary was introduced via an on - resin fukuyama synthesis as described in example 3 , using the reaction sequence summarized in scheme 12 , wherein ala - phe - leu - pro - ala is seq id no : 19 . cyclisation of the n - ethanethiol derivative 1 yielded only the monocyclic product 2 ( 45 % isolated yield ), as determined by mass spectral analysis ( correct molecular weight and isotope distribution ). no dimeric or other oligomeric products were found in the crude reaction mixture . the monocyclic product has the thioester structure , as confirmed by saponification of the monocyclic product 2 in nh 4 hco 3 which generated disulfides 3 of the linear peptide amides and acids . attempts to force ring contraction by heating ( 65 ° c .) the isolated ester in organic solvents ( dmf , dioxane ) in the presence of base ( diea , dbu ), or heating in aqueous buffers ( ph 4 - 8 ) failed . the ester either remained unchanged or hydrolysed to the linear peptide . this failure to ring contract is in our view due to the low reactivity of the alkylthioester towards secondary amines rather than a constrained transition state geometry . we decided to examine the 2 - hydroxynitrobenzaldehydes in a ring contraction approach , as initial cyclisation would generate a more reactive nitrophenylester with significantly improved acyl transfer kinetics . the peptide 4a was synthesised as described in example 4 . cyclisation of peptide 4a under standard conditions initially yielded two monocyclic products , as well as significant amounts of a side product 6a ( mr , 812 da ), caused by reaction of the phenol functionality with excess bop in the reaction mixture ( scheme 13 , a ). by adjusting the amount of activating reagent and base , formation of this side product was completely avoided . the reaction conditions were further optimised by altering the temperature and amount of base after an initial cyclisation period and monitoring the formation of monocyclic products by lc / ms analysis . the best results were obtained when after 3 h of reaction ( 1 mm in dmf , leg bop , 2 eq diea , rt ) excess diea ( 10 eq ) was added and the mixture left standing for 24 h or heated to 65 ° c . for 1 hour . scheme 13 : cyclisation of auxiliary containing peptides 4 , 5 ( a ) and formation of the target cyclic peptides 10 , 11 ( b ); i ) 3 eq . bop / 5 eq . diea , 3 h at rt ; ii ) 1 eq . bop / 2 eq . diea , 3 h rt ; 10 eq . diea , 12 h rt or 1 h at 65 ° c . ; iii ) hv ( 366 nm ), wherein ala - phe - leu - pro - ala is seq id no : 19 and phe - leu - pro - ala - ala is seq id no : 31 . the hplc profile of the crude product is depicted in fig2 b . the main product ( 50 % isolated yield ) was unambiguously characterised by nmr , es - ms and chiral amino acid analysis as the all - l target monocyclic product 10a . a 1 h nmr absorption at 11 . 5 ppm confirmed that the product contained the free hydroxy substituent , and thus did not have the ester structure but rather the target cyclic amide structure . further , a small amount of the c - terminally racemised product 10b was also isolated ( see fig2 b ). a chiral amino acid analysis of the product displayed the presence of a d - ala residue . in an attempt to isolate the intermediate cyclic ester 8a , the reaction mixture was analysed after the initial 3 h cyclisation period by hplc ( fig2 a ) and lc / ms . the mixture contained linear peptide and monocyclic products 10a and 10b , but no monocyclic ester was found . the p - nitrophenyl ester presumably hydrolyses in the aqueous work - up to the linear peptide . as the 5 - nitro - 2 - hydroxybenzyl auxiliary is not readily removed after cyclisation , we examined the 6 - nitro - 2 - hydroxybenzyl auxiliary peptide 5a towards cyclisation . the ortho - nitro substituent , while maintaining a similar activation effect on the ring contraction of the cyclic intermediate 9a compared to 8a , has the added benefit that it should render the auxiliary photolabile . the linear peptide 5a was synthesised and treated as described above for the 5 - nitro - 2 - hydroxy derivative . thus cyclisation ( at 1 mm in dmf , 1 eq . bop / 2 eq . diea ) was performed at rt for 3 h , followed by addition of excess diea ( 10 eq ) and heating to 65 ° c . for 1 hour . the major product was isolated in 39 % yield , and characterised by nmr and chiral amino acid analysis as the all - l cyclo - pentapeptide 11a . a small amount of the c - terminal racemised cyclic product 11b ( containing a d - ala ) was also isolated . similarly n -( 6 - nitro - 2 - hydroxybenzyl ) phe - leu - pro - ala - ala ( seq id no : 11 ) 5c was assembled and cyclised as above . the all - l cyclo pentapeptide 11c was isolated in 45 % yield . removal of the auxiliary . cyclic peptide 11a was then subjected to photolysis at 366 nm , using a standard uv lamp , in a range of solvent conditions . in most solvents ( meoh , meoh / acoh , thf / acoh , dioxane ) the nitrobenzyl substituent on the backbone nitrogen is readily removed to generate the target cyclic peptide 12a ( scheme 14 , b ). fig3 illustrates the clean and efficient conversion ( 11a to 12a ). the cyclic product was characterised by chiral amino acid analysis and 1 h nmr . the spectral data were in good agreement with the reported data . furthermore , an independent sample of cyclic peptide , prepared from the cyclisation of phe - leu - pro - ala - ala ( seq id no : 31 ) according to schmidt et al ( 1997 ) coeluted with the product obtained from photolysis . the same product 12a was obtained from photolysis of the regio analogue 11c . the racemised cyclic product 11b was photolysed , and similarly produced the unsubstituted d - ala containing product 12b , which coeluted with an independently synthesised sample . cyclisation experiments . cyclisation of auxiliary - containing peptides 4 and 5 : 1 equivalent of bop and 2 equivalents of diea in dmf were added to a 1 mm solution of the linear peptide in dmf and stirred for 3 h at rt . 10 equivalents of diea was then added and the solution heated at 65 ° for 1 h . dmf was removed in vacuo and the crude product dissolved in acetonitrile / water ( 1 : 1 ) and purified by rp - hplc . cyclisation of other linear peptides : cyclisations were performed using a 1 mm solution of linear peptide in dmf . 3 equivalents of bop and 5 equivalents of diea were added and the solution stirred for 3 h at rt . work - up was as described above . cyclo -[ s —( ch 2 ) 2 - ala - phe - leu - pro - ala ] ( seq id no : 12 ) 2 . cyclisation of hs —( ch 2 ) 2 - ala - phe - leu - pro - ala ( seq id no : 13 ) 1 ( 10 mg of the tfa salt , 0 . 014 mmol ) produced the monocyclic thioester 2 ( 3 . 4 mg , 45 % yield ): mr : 559 . 3 , calcd for c 28 h 41 n 5 o 5 s : 559 . 3 . the thioester was hydrolysed using aqueous ammonium bicarbonate buffer ( 0 . 1 m , ph 8 , 6 h at 60 ° c .) to form the c - terminal amides and acids . under the mild base conditions these thiol - products oxidised to the disulfides 3 which were characterised by es - ms . [ s —( ch 2 ) 2 — nh — ch ( ch 3 )— co - phe - leu - pro - ala - nh 2 ] 2 ( seq id no : 40 ) mr : 1150 . 8 , calcd for c 56 h 86 n 12 o 10 s 2 : 1150 . 6 , [ s —( ch 2 ) 2 — nh — ch ( ch 3 )— co - phe - leu - pro - ala - nh 2 ]— s —( ch 2 ) 2 — nh — ch ( ch 3 )— co - phe - leu - pro - ala - oh ( seq id no : 14 ) mr : 1151 . 8 , calcd for c 56 h 85 n 11 o 11 s 2 : 1151 . 6 , [ s —( ch 2 ) 2 — nh — ch ( ch 3 )— co - phe - leu - pro - ala - oh ] 2 ( seq id no : 41 ) mr : 1152 . 8 , calcd for c 56 h 84 n 10 o 12 s 2 : 1152 . 6 . cyclo -[ n -( 5 - nitro - 2 - hydroxybenzyl )- ala - phe - leu - pro - ala ] ( seq id no : 42 ) ( 10a ). cyclisation of n -( 5 - nitro - 2 - hydroxybenzyl )- ala - phe - leu - pro - ala ( seq id no : 15 ) 4a ( 30 mg of the tfa salt , 0 . 038 mmol ), produced 10a ( 12 . 5 mg , 0 . 019 mmol ) in 51 % yield : es - ms mr 650 . 2 , calcd for c 33 h 42 n 6 o 8 , 650 . 3 ( monoisotopic ). 1 h nmr ( 500 mhz , dmso - d 6 , ppm ) δ 11 . 5 ( s , 1h , oh ), 8 . 40 ( d , 1h , nh leu ), 8 . 02 ( dxd , 1h , h - ar ), 7 . 70 ( d , 1h , h - ar ), 7 . 4 ( d , 1h , hn phe ), 7 . 20 - 7 . 30 ( m , 5h , h - phe ), 6 . 99 ( d , 1h , h - ar ), 6 . 54 ( d , 1h , h — n ala ), 5 . 00 ( s , 1h , archhn —), 4 . 91 ( m , 1h , α - ala 5 ), 4 . 75 ( q , 1h , α - ala 1 ), 4 . 59 ( m , 1h , α - phe ), 4 . 50 ( m , 1h , α - leu ), 4 . 27 ( t , 1h , α - pro ), 3 . 88 ( d , 1h , archhn —), 3 . 62 ( m , 1h , δ - pro ), 3 . 37 ( m , 1h , δ - pro ), 2 . 97 ( m , 1h , β - phe ), 2 . 82 ( m , 1h , β - phe ), 2 . 04 ( m , 2h , β - pro ), 1 . 88 ( m , 1h , γ - pro ), 1 . 73 ( m , 1h , β - leu ), 1 . 65 ( m , 1h , γ - pro ), 1 . 44 ( m , 1h , γ - leu ), 1 . 33 ( m , 1h , β - leu ), 1 . 24 ( d , 3h , β - ala 5 ), 0 . 91 ( d , 3h , β - ala 1 ), 0 . 85 ( m , 6h , δ - leu ). 13 c nmr ( 75 mhz , dmso - d 6 , ppm ) δ 172 . 61 , 170 . 34 , 170 . 07 , 169 . 95 , 169 . 47 , 160 . 40 , 139 . 73 , 136 . 88 , 129 . 31 , 128 . 14 , 126 . 50 , 125 . 72 , 124 . 21 , 122 . 65 , 115 . 00 , 61 . 04 , 56 . 50 , 55 . 74 , 48 . 70 , 46 . 31 , 44 . 34 , 41 . 37 , 38 . 28 , 31 . 30 , 24 . 20 , 22 . 81 , 22 . 68 , 21 . 17 , 18 . 97 , 15 . 35 . cyclo -[ n -( 6 - nitro - 2 - hydroxybenzyl )- ala - phe - leu - pro - ala ] ( seq id no : 16 ) ( 11a ). from cyclisation of n -( 6 - nitro - 2 - hydroxybenzyl )- ala - phe - leu - pro - ala ( seq id no : 43 ) 5a ( 20 mg of the tfa salt , 0 . 025 mmol ), 11a ( 6 . 5 mg , 0 . 010 mmol ) was obtained in 39 % yield : es - ms mr 650 . 6 , calcd for c 33 h 42 n 6 o 8 : 650 . 3 ( monoisotopic ). 13 c nmr ( 75mhz , cd 3 od , ppm ) δ 178 . 07 , 176 . 95 , 174 . 54 , 174 . 32 , 173 . 72 , 159 . 11 , 153 . 19 , 140 . 41 , 131 . 99 , 129 . 96 , 129 . 54 , 127 . 57 , 121 . 18 , 116 . 57 , 62 . 75 , 60 . 67 , 58 . 55 , 54 . 05 , 51 . 15 , 44 . 54 , 43 . 41 , 34 . 85 , 33 . 67 , 25 . 03 , 24 . 13 , 22 . 30 , 21 . 31 , 15 . 49 , 13 . 89 . cyclo -[ n -( 6 - nitro - 2 - hydroxybenzyl )- phe - leu - pro - ala - ala ] ( seq id no : 17 ) ( 11c ). from cyclisation of the n -( 6 - nitro - 2 - hydroxybenzyl )- phe - leu - pro - ala - ala ( seq id no : 18 ) ( 20 mg of the tfa salt , 0 . 025 mmol ), 11a ( 7 . 3 mg , 0 . 011 mmol ) was obtained in 44 % yield : es - ms mr 650 . 2 , calcd for c 33 h 42 n 6 o 8 : 650 . 3 ( monoisotopic ). 13 c nmr ( 75 mhz , dmso - d6 , ppm ) δ 171 . 43 , 171 . 00 , 169 . 46 , 167 . 56 , 156 . 65 , 138 . 43 , 129 . 24 , 129 . 05 , 128 . 32 , 128 . 18 , 126 . 08 , 119 . 50 , 115 . 87 , 114 . 60 , 62 . 18 , 60 . 69 , 51 . 07 , 49 . 38 , 46 . 57 , 45 . 46 , 41 . 54 , 38 . 17 , 33 . 65 , 31 . 43 , 24 . 37 , 22 . 73 , 22 . 32 , 21 . 06 , 17 . 87 , 16 . 92 . cyclo -[ ala - phe - leu - pro - ala ] ( seq id no : 10 ) ( 12a ). a ) cyclo -[ n -( 6 - nitro - 2 - hydroxybenzyl )- ala - phe - leu - pro - ala ] ( seq id no : 16 ) ( 1 mm meoh ) was purged with nitrogen for 30 minutes and then photolysed with a standard laboratory uv lamp ( 366 nm , 0 . 25 a ) for three hours . the meoh was evaporated and residue dissolved in 50 % buffer b and the solution loaded directly onto a vydac c18 column ( preparative ) for hplc purification . cyclo -[ ala - phe - leu - pro - ala ] ( seq id no : 10 ) was isolated in 52 % yield . the product coeluted with a independently synthesised sample . es - ms mr 499 . 4 , calcd for c 26 h 37 n 5 o 5 , 499 . 3 ( monoisotopic ). b ) photolysis of purified cyclo -[ n -( 6 - nitro - 2 - hydroxybenzyl )- phe - leu - pro - ala - ala ] ( seq id no : 17 ) was performed as described above . cyclo -[ phe - leu - pro - ala - ala ] ( seq id no : 20 ) was isolated in 28 % yield . the product coeluted with a independently synthesised sample . es - ms mr 499 . 1 , calcd for c 26 h 37 n 5 o 5 , 499 . 3 ( monoisotopic ). application of the photolabile auxiliary for the cyclisation of an all - l - tetrapeptide , tyr - arg - phe - gly ( seq id no : 21 ), via a ring contraction approach we decided to investigate the feasibility of our auxiliary approach for the synthesis of a more constrained all - l cyclo tetrapeptide . standard cyclisation of the linear peptide tyr - arg - phe - gly ( seq id no : 21 ) yields cyclic monomer / cyclic dimer / cyclic trimer in a ratio of 2 / 8 / 3 . cyclisation of ( hnb ) tyr - arg - phe - gly ( seq id no : 22 ) was performed as described before , but heating ( 65 ° c . after diea addition ) was continued for 20 hours ( instead of 1 h ). the product cyclo -[( hnb ) tyr - arg phe - gly ] ( seq id no : 23 ) was isolated in 40 % yield . photolysis of this product in meoh / acoh was slow and yielded impure cyclo [ tyr - arg - phe - gly ] ( seq id no : 24 ), whereas photolysis in thf , dmf or dioxane was significantly faster ( complete in hour ). the cyclo -[ tyr - arg - phe - gly ] ( seq id no : 24 ) was isolated in 41 % yield ( photol . step ). in this experiment we illustrate how we bring this auxiliary approach into practice for performing native ligation . two ligation sites were examined , ie . a non - hindered site , gly - gly , and a more hindered site , phe - ala . the oxyethanethiol auxiliary approach has been applied to these ligation sites ( canne et al , 1996 ), and was only successful in the first , non - hindered , case . the first peptide segments ( peptide 1 ; gagpa is seq id no : 26 and aarht is seq id no : 29 ) are assembled using standard in situ neutralisation protocols and the auxiliary is introduced as described in examples 1 to 5 . standard hf cleavage and side chain deprotection provides the first unprotected peptide segment . the second peptide segments ( peptide 2 ; lyrag is seq id no : 25 and lyraf is seq id no : 28 ), containing a thiophenylester at the c - terminus , are synthesised as described before ( canne et al , 1996 ). to optimise the ligation conditions the following experiments are then performed : peptide 1 and peptide 2 are dissolved in dmf at 1 mm , 10 mm and 100 mm concentration and 2 or 5 equivalents of diea added . progress of the reaction is monitored for each experiment by hplc and lcms analysis at different time intervals . several other solvent systems are tested , such as dmso / diea , and aqueous buffers ( ph ranging from 4 to 8 ) ( no diea ). the products are lyraggagpa ( seq id no : 27 ) and lyrafaarht ( seq id no : 30 ). in this experiment we illustrate how we accommodate photolabile backbone linking using this auxiliary approach for the solid phase synthesis of cyclic peptides . cyclo - flpaa is seq id no : 20 . 2 , 3 - dihydroxy - 6 - nitrobenzaldehyde ( perez et , al , 1992 ) is treated with 1 equivalent bromovaleric acid and 1 equivalent khco 3 in acetone . the resulting acid is linked to aminomethylated polystyrene . alanine allyl ester is attached to the resin by reductive amination and the resulting secondary amine acylated with boc - ala - oh as described in example 6 . the linear peptide ( phe - leu - pro - ala - ala ) ( seq id no : 31 ) is further assembled using in situ neutralisation protocols . the n - terminus is deprotected with tfa and the c - terminal allyl protection group removed using pd [ p ( ph ) 3 ] 4 as described . the cyclisation is then performed with bop / diea in dmf and the product cleaved from the resin by photolysis . it will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding , various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification . references cited herein are listed on the following pages , and are incorporated herein by this reference . albericio , f . and carpino , l . a . methods in enzymology , 1997 289 104 - 125 alewood , p ., alewood , d ., miranda , l ., love , s ., meutermans , w . and wilson , d . methods in enzymology , 1997 289 , 14 - 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