Patent Application: US-24644102-A

Abstract:
disclosed are β - peptides and β - peptide conjugates that are capable of diffusing or otherwise being transported across the cell membranes of living cells . the β - peptides contain at least six β - amino acid residues , at leastsix of which are preferably β 3 - homoarginine residues . it has been found that when pharmacologically - active agents are conjugated to these types of β - peptides , the resulting conjugates are also capable of diffusing or otherwise being transported across the cell membranes of living cells , including mammalian cells .

Description:
the following abbreviations are used throughout the specification and claims . unless specifically defined to the contrary , all other terms have their standard accepted meanings . all of the following compounds can be purchased commercially from aldrich chemical company , milwaukee , wis ., usa , as well as other national and international suppliers : as used herein , the terms “ β - amino acid ” and “ β - amino acid residue ” refer to any and all natural and unnatural β - amino acids and their respective residues ( i . e ., the form of the amino acid when incorporated into a polypeptide molecule ), without limitation . specifically included are those β - amino acids described in u . s . pat . no . 6 , 060 , 585 , issued may 9 , 2000 , to gellman et al . and incorporated herein by reference . a “ β - peptide ” is a polyamide comprising β - amino acid residues . the term “ cyclically - constrained ,” when applied to β - amino acids and residues , designates that the α and β backbone carbon atoms of each β - amino acid or residue are incorporated into a c 3 - c 10 cycloalkyl , cycloalkenyl , aryl , or hetercyclic ring . all stereochemical configurations ( single enantiomers , single diastereomers , mixtures thereof , and racemates thereof ) of the compounds described herein are encompassed within the scope of the invention . in the preferred embodiments , all of the residues share the same absolute configuration ( either r or s ) about the asymmetric ring carbon in the position p to the exocyclic carbonyl carbon , when the conjugate contains residues that are cyclic imino carboxylic acids . as used in the specification and the claims , the word “ independently ,” when referring to the nature of a variable substituent , explicitly means that each appearance of the defined substituent within a molecule can be different . thus , for example , in a molecule according to the present invention such as a - x 3 - z 3 - b ( wherein a is hydrogen , and b is hydroxy ), each appearance of x and each appearance of z can vary independently within the molecule . thus , according to this explicit definition , the molecule a - x 3 - z 3 - b explicitly encompasses the molecule a - x ′- x ″- x ′″- z ′- z ″- z ′″- b , where x ′ may the same as or different from x ″, and x ″ may be the same as or different from x ′″. likewise , z ′ may the same as or different from z ″, and z ″ may be the same as or different from z ′″. as used herein , the terms “ amino - terminus protecting group ” and “ carboxy - terminus protecting group ” refer to any chemical moiety capable of addition to and ( optionally ) removal from a reactive site ( an amino group and a carboxy group , respectively , in this instance ) to allow manipulation of a chemical entity at sites other than the reactive site . protecting groups , and the manner in which they are introduced and removed are described , for example , in “ protective groups in organic chemistry ,” plenum press , london , n . y . 1973 ; and in “ methoden der organischen chemie ,” houben - weyl , 4th edition , vol . 15 / 1 , georg - thieme - verlag , stuttgart 1974 ; and in theodora w . greene , “ protective groups in organic synthesis ,” john wiley & amp ; sons , new york 1981 . a characteristic of many protecting groups is that they can be removed readily , i . e ., without the occurrence of undesired secondary reactions , for example by solvolysis , reduction , photolysis or alternatively under physiological conditions . a host of protecting groups are known in the art . an illustrative , non - limiting list of protecting groups includes methyl , formyl , ethyl , acetyl , t - butyl , benzyl , trifluoroacetyl , t - butoxycarbonyl , benzoyl , 4 - methylbenzyl , benzyloxymethyl , 4 - nitrophenyl , benzyloxycarbonyl , 2 - nitrobenzoyl , 2 - nitrophenylsulphenyl , 4 - toluenesulphonyl , pentafluorophenyl , diphenylmethyl , 2 - chlorobenzyloxycarbonyl , 2 , 4 , 5 - trichlorophenyl , 2 - bromobenzyloxycarbonyl , 9 - fluorenylmethyloxycarbonyl , triphenylmethyl , and 2 , 2 , 5 , 7 , 8 - pentamethyl - chroman - 6 - sulphonyl . the terms “ amino - terminus protecting group ” and “ carboxy - terminus protecting group ” as used herein are explicitly synonymous with such terms as “ n - terminal capping group ” and “ c - terminal capping group ,” respectively . a host of suitable protecting and capping groups , in addition to those described above , are known in the art . for discussions of various different types of amino - and carboxy - protecting groups , see , for example , u . s . pat . no . 5 , 221 , 736 ( issued jun . 22 , 1993 ); u . s . pat . no . 5 , 256 , 549 ( issued oct . 26 , 1993 ); u . s . pat . no . 5 , 049 , 656 ( issued sep . 17 , 1991 ); and u . s . pat . no . 5 , 521 , 184 ( issued may 28 , 1996 ). regarding salts of the subject compounds , compounds having at least one basic group or at least one basic radical , for example a free amino group , a free guanidino group , a pyrazinyl radical , or a pyridyl radical , may form acid addition salts . thus , the invention encompasses acid addition salts of the subject compounds with ( for example ) inorganic acids , such as hydrochloric acid , sulfuric acid or a phosphoric acid , or with suitable organic carboxylic or sulfonic acids , for example aliphatic mono - or di - carboxylic acids , such as trifluoroacetic acid , acetic acid , propionic acid , glycolic acid , succinic acid , maleic acid , fumaric acid , hydroxymaleic acid , malic acid , tartaric acid , citric acid or oxalic acid , or amino acids such as arginine or lysine , aromatic carboxylic acids , such as benzoic acid , 2 - phenoxy - benzoic acid , 2 - acetoxybenzoic acid , salicylic acid , 4 - aminosalicylic acid , aromatic - aliphatic carboxylic acids , such as mandelic acid or cinnamic acid , heteroaromatic carboxylic acids , such as nicotinic acid or isonicotinic acid , aliphatic sulfonic acids , such as methane -, ethane - or 2 - hydroxyethane - sulfonic acid , or aromatic sulfonic acids , for example benzene -, p - toluene - or naphthalene - 2 - sulfonic acid . when several basic groups are present mono - or poly - acid addition salts may be formed . as used herein , the terms alkyl , alkenyl , and alkynyl explicitly encompass linear , branched and / or cyclic moieties , including mono - and bicyclic moieties . in the case of disubstituted amine , amide and carboxamide moieties ( e . g ., a di - c 1 - c 6 - alkyl - substituted amine ), the disubstitution explicitly encompasses substitution patterns wherein the nitrogen atom defines part of a heterocyclic ring . when the subject compounds have acidic groups , for example a free carboxy group , the invention encompasses metal and ammonium salts , such as alkali metal or alkaline earth metal salts , for example sodium , potassium , magnesium or calcium salts , or ammonium salts with ammonia or suitable organic amines , such as tertiary monoamines , for example triethylamine or tri -( 2 - hydroxyethyl )- amine , or heterocyclic bases , for example n - ethylpiperidine or n , n ′- dimethyl - piperazine . compounds of the present invention having both acidic and basic groups can form internal salts . the salts may be pharmaceutically - acceptable salts or pharmaceutically - unacceptable salts . the cyclically - constrained β - amino acid residues that can be utilized in the present invention include those disclosed in u . s . pat . no . 6 , 060 , 585 , issued may 9 , 2000 , to gellman et al ., and incorporated herein by reference . additionally , β - amino acid residues such as those disclosed in co - pending application ser . no . 09 / 502 , 829 , to gellman et al . ( incorporated herein ), may also be used in the present invention . further still , cyclic imino carboxylic acids and gem - di - substituted cyclic imino carboxylic acids ( both of which are a type of cyclically - constrained β - amino acid ) can also be used in the invention . preferably , these residues take the form of the individual residues disclosed in co - pending application ser . no 09 / 592 , 769 , to gellman et al ., incorporated herein by reference . further still , these β - residues may also take the form of the gem - di - substituted cyclic imino acids disclosed in co - pending application ser . no . 09 / 883 , 579 , to gellman & amp ; huck , incorporated herein by reference . general : melting points are uncorrected . ch 2 cl 2 was freshly distilled from cah 2 under n 2 . dmf was distilled under reduced pressure from ninhydrin and stored over 4 å molecular sieves . triethylamine was distilled from cah 2 before use . other solvents and reagents were used as obtained from commercial suppliers . for boc removal , 4 m hcl in dioxane from was used . column chromatography was carried out by using low air pressure ( typically 6 psi ) with 230 - 400 mesh silica gel 60 . routine 1 h - nmr spectra were obtained on a bruker ac - 300 and are referenced to residual protonated nmr solvent . routine 13 c - nmr spectra were obtained on a bruker ac - 300 and are referenced to the nmr solvent . high resolution electron impact mass spectroscopy was performed on a kratos ms - 80rfa spectrometer with ds55 / ds90 . far uv circular dichroism ( cd ): data were obtained on a jasco j - 715 instrument at 20 ° c . in all cd plots contained herein , the mean residue ellipticity is presented on the vertical axis . presenting the mean residue ellipticity is a standard practice in peptide chemistry wherein the intensity of each cd spectrum is normalized for the number of amide chromophores in the peptide backbone . consequently , when the intensities of the maximum and minimum peaks characteristic of secondary structure formation increase with increasing chain length , this change represents an increase in the population of the secondary structure , rather than simply an increase in the number of chromophores present in each molecule . solid - phase and solution - phase polypeptide synthesis : construction of polypeptides using any type of β - amino acid residue can be accomplished using conventional and widely recognized solid - phase or solution - phase synthesis . very briefly , in solid - phase synthesis , the desired c - terminal amino acid residue is linked to a polystyrene support as a benzyl ester . the amino group of each subsequent amino acid to be added to the n - terminus of the growing peptide chain is protected with boc , fmoc , or another suitable protecting group . likewise , the carboxylic acid group of each subsequent amino acid to be added to the chain is activated with dcc and reacted so that the n - terminus of the growing chain always bears a removable protecting group . the process is repeated ( with much rinsing of the beads between each step ) until the desired polypeptide is completed . in the classic route , the n - terminus of the growing chain is protected with a boc group , which is removed using trifluoracetic acid , leaving behind a protonated amino group . triethylamine is used to remove the proton from the n - terminus of the chain , leaving a free amino group , which is then reacted with the activated carboxylic acid group from a hew protected amino acid . when the desired chain length is reached , a strong acid , such as hydrogen bromide in trifluoracetic acid , is used to both cleave the c - terminus from the polystyrene support and to remove the n - terminus protecting group . solid - phase peptide synthesis is widely employed and well known . consequently , it will not be described in any further detail here . see , for example , “ peptide synthesis , structures , and applications ”© 1995 by academic press . conventional solution - phase peptide synthesis can also be used with equal success . β - amino acid residues : cyclically - constrained β - amino acid residues that can be used in the present invention are exemplified by compounds such as 2 - aminocyclohexanecarboxylic acid ( achc ) and 2 - aminocyclopentanecarboxylic acid ( acpc ). these two structures are shown below : briefly , for unsubstituted β - amino acids and β - amino acids containing one or two acyclic substituents on the carbon adjacent to the amino group in the product β - peptide , the arndt - eistert homologation reaction can be used , see reaction 1 . see also seebach et al . ( 1996 ) helv . chim . acta 79 : 913 . a distinct advantage to this route is that the starting materials , α - amino acids , are readily available commercially in enantiomerically pure form . the arndt - eistert reaction , however , cannot be used to synthesize β - amino acids having rings in the backbone . the arndt - eistert homologation reaction proceeds via a wolff rearrangement of a diazoketone , as shown in reaction 1 : ( pg ) designates a protecting group such as ( t - butoxy ) carbonyl ( boc ) or an adjacent β - amino residue , r 1 and r 2 are aliphatic substituents . the arndt - eistert homologation procedure can also be used to make β - amino acids having an α - position substituent via alkylation of the intermediate α - diazoketone . see yang et al . ( 2000 ), org . lett . 2 : 2177 : 2179 . β - amino acids containing an unsubstituted cycloalkyl moiety involving the α and β carbons are synthesized using literature methods . see , for example , nohira et al . ( 1970 ) bull . chem . soc . jpn . 43 : 2230 ; herradon and seebach ( 1989 ) helv . chim . acta 72 : 690 - 714 ; and tilley et al . ( 1992 ) j . med . chem . 35 : 3774 - 3783 , all three of which are incorporated herein by reference . see also gellman et al ., u . s . pat . no . 6 , 060 , 585 , issued may 9 , 2000 , also incorporated herein by reference . in particular , the cyclohexyl - containing β - amino acids can be synthesized via reaction 2 : ( 1r , 6s )- 6 - methoxycarbonyl - 3 - cyclohexene - 1 - carboxylic acid ( 23 ): 4600 u of ple was suspended in ph 8 . 01 aqueous buffer solution ( 0 . 17 m kh 2 po 4 ). the diester 22 ( 10 . 1 g , 0 . 05 mol ) was dissolved in 30 ml of acetone and added to the buffer solution . reaction was allowed to stir at rt overnight . the enzyme was filtered off through a well - packed celite pad , the solution was then acidified to ph 1 with 1m hcl and the product was extracted with ethyl acetate ( 5 × 400 ml ). the combined organic extracts were dried over anhydrous magnesium sulfate and concentrated to yield 9 . 00 g yellow oil . product taken on without further purification . methyl ( 1s , 6r )- 6 - benzyloxycarbonylaminocyclohex - 3 - ene carboxylate ( 24 ): ethylchloroforamate ( 4 ml , 0 . 042 mol ) was added to a mixture of 23 ( 5 . 14 g , 0 . 028 mol ) and triethylamine ( 6 ml , 0 . 043 mol ) in acetone ( 100 ml ) at 0 ° c . and vigorously stirred for 10 min . an aqueous solution of nan 3 ( 3 . 04 g , 0 . 047 mol , in 25 ml water ) was added in one portion . the resulting mixture was stirred for 30 min at 0 ° c . the reaction mixture was diluted with water and extracted with diethyl ether . the organic extracts were dried over anhydrous magnesium sulfate and concentrated without heat to yield a viscous yellow liquid . the liquid was dissolved in 100 ml of benzene and refluxed under nitrogen atmosphere for 30 min . benzyl alcohol ( 12 ml , 0 . 116 mol ) was added and solution was refluxed for an additional 16 h . the reaction was cooled to rt and concentrated to yield 17 . 12 g of a yellow liquid ( mixture of benzyl alcohol and desired product in a 5 . 4 : 1 ratio , respectively by 1 h nmr , ˜ 5 . 67 g product ). mixture taken on without further purification . methyl ( 1s , 6r )- 6 - tert - butoxycarbonylaminocyclohexane carboxylate ( 25 ): the yellow oil from the previous reaction , which contains compound 24 ( 5 . 6 g , 0 . 020 mol ) and benzyl alcohol , was dissolved in methanol . 0 . 525 g of 10 % pd on carbon was added to the methanol solution , and the heterogenous mixture was placed under 50 psi h 2 and shaken at rt for 24 h . the mixture was filtered through celite , and the filtrate was concentrated to yield 13 . 74 g of dark golden yellow liquid . 25 ml of 1m hcl was added to the filtrate , and the benzyl alcohol was extracted with diethyl ether ( 3 × 25 ml ). the ph of the aqueous solution was adjusted to 9 using k 2 co 3 . 25 ml of dioxane and boc 2 o ( 5 g , 0 . 023 mol ) were added to the solution , and the reaction was stirred at rt for 20 h . 15 ml of water was added and the solution was extracted with ethyl acetate ( 3 × 50 ml ). the combined organic extracts were dried over anhydrous magnesium sulfate and concentrated . residue was purified via column chromatography ( sio 2 , eluting with 6 : 1 hex : etoac ), to yield 2 . 00 g viscous clear oil . methyl ( 1r , 6r )- 6 - tert - butoxycarbonylaminocyclohexane carboxylate ( 26 ): sodium metal ( 0 . 14 g , 6 . 1 mmol ) was placed into a flame dried flask under nitrogen atmosphere and cooled to 0 ° c . 10 ml of freshly distilled methanol was added and the mixture stirred until all the sodium dissolved . an amount of 25 ( 2 . 00 g , 7 . 7 mmol ) was dissolved in 10 ml of freshly distilled methanol and transferred to naome solution via cannula . the solution was refluxed under nitrogen for 5 . 5 h , cooled to rt and acidified with 0 . 5 m aqueous 0 . 5 m ammonium chloride ( 18 ml , 9 mmol ). the methanol was removed under reduced pressure , and the resulting solid collected by filtration to yield 1 . 27 g of desired product . β - amino acids containing a substituted cycloalkyl moiety were synthesized using the following illustrative protocol , the first four steps of which are described in kobayashi et al . ( 1990 ) chem . pharm . bull . ( 1990 ) 38 : 350 . the remaining steps to yield a cyclohexyl ring having two differentially protected amino substituents were developed in furtherance of the present invention and have not heretofore been described in the literature and are shown in reaction 3 : as depicted in reaction 3 , the 4 - position amino substituent is protected by a boc group and the 1 - position amino substituent is protected by a cbz group . the starting material is available commercially ( aldrich chemical co ., milwaukee , wis .). synthesis of β - amino acids containing a heterocylic ring moiety encompassing the α and β carbons were synthesized using reactions 4 and 5 , below . reaction 4 details an illustrative synthesis of a β - proline wherein the exocyclic amino substituent is in the 3 - position relative to the ring nitrogen . compound 42 : tap water ( 200 ml ) and baker &# 39 ; s yeast ( 25 g ) were mixed , and were shaken on an orbital shaker for 1 hour . compound 41 ( 1 . 0 g ) was then added . the mixture was shaken at room temperature for 24 hours . the mixture was filtered through a bed of celite . the celite was washed with water ( 20 ml ). the filtrate was extracted with diethyl ether ( 5 × 100 ml ). the extracts were washed with water ( 2 × 50 ml ), dried over mgso 4 , and concentrated to yield a slightly yellow oil . the crude product was purified by column chromatography with ethyl acetate / hexane ( 1 / 1 , v / v ) as eluent to give a colorless oil ( 0 . 5 g ) in 50 % yield . starting material : blake , j . et al j . am . chem . soc . 1964 , 86 5293 . yeast reduction : cooper , j . et al j . chem . soc . perkin trans . 1 1993 , 1313 . compound 43 : compound 42 ( 228 mg ) and ph 3 p ( 346 mg ) were dissolved in benzene ( anhydrous , 4 ml ) under nitrogen . hn 3 ( 1 . 64 m in benzene , 0 . 8 ml ) was then added . a solution of diethyl azodicarboxylate ( 0 . 18 ml ) in benzene ( 1 . 0 ml ) was subsequently introduced via syringe over 5 minutes . the reaction mixture turned cloudy towards the end of the addition . the reaction mixture was stirred under nitrogen at room temperature for 3 . 0 hours . the reaction mixture was then taken up in ethyl acetate ( 50 ml ), washed with 1n naoh ( 10 ml ), saturated nahco 3 ( 10 ml ), and finally dilute brine ( 5 ml ). the organic extract was dried over mgso 4 , and concentrated to give a slightly yellow oil . the crude oil was purified by column chromatography with ethyl acetate / hexane ( 1 / 1 , v / v ) as eluent to afford a colorless oil ( 190 mg ) in 76 % yield . compound 44 : compound 43 ( 1 . 1 g ) was dissolved in methanol ( 50 ml ). sncl 2 ( 2 . 2 g ) was then added . the mixture was stirred at room temperature for 30 hours . the methanol was then removed under reduced pressure . the residue was dissolved in methylene chloride ( 50 ml ). the resulting cloudy solution was filtered through celite . the methylene chloride was then removed under reduced pressure . the residual white solid was dissolved in acetone / water ( 2 / 1 , v / v , 50 ml ). nahco 3 ( 3 . 3 g ) was added , followed by cbz - osu ( 1 . 16 g ). the reaction mixture was stirred at room temperature for 24 hours . water ( 50 ml ) was added . the acetone was removed under reduced pressure . the aqueous mixture was extracted with ethyl acetate ( 3 × 100 ml ). the extracts were washed with dilute brine ( 30 ml ), dried over mgso 4 , and concentrated to give a colorless oil . the crude product was purified by column chromatography with ethyl acetate / hexane ( 3 / 7 , v / v ) as eluent to give the clean product as a colorless oil ( 1 . 35 g ) in 89 % yield . compound 45 : compound 44 ( 1 . 35 g ) was dissolved in methanol / water ( 3 / 1 , v / v , 80 ml ), cooled to 0 ° c . lioh . h 2 o ( 1 . 68 g ) was added . the mixture was stirred at 0 ° c . for 24 hours , by which time tlc indicated that the hydrolysis was complete . saturated ammonium hydroxide ( 20 ml ) was added . the methanol was removed under reduced pressure . the aqueous was washed with diethyl ether ( 50 ml ), acidified with 1n hcl to ph 3 , extracted with methylene chloride ( 3 × 150 ml ). the extracts were washed with dilute brine ( 50 ml ), dried over mgso 4 , concentrated to give a sticky colorless residue ( 1 . 25 g , 99 %), which was used directly without further purification . compound 46 : compound 45 ( 1 . 25 g ) was dissolved in methanol ( 50 ml ) in a hydrogenation flask . 5 % palladium on activated carbon ( 190 mg ) was added . the flask was pressurized with hydrogen to 35 psi , rocked at room temperature for 7 hours , by which time tlc indicated that the hydrogenolysis was complete . the pd / c was removed by filtration . the filtrate was concentrated to give a white solid . the white solid was dissolved in acetone / water ( 2 / 1 , v / v , 70 ml ), cooled to 0 ° c . nahco 3 ( 1 . 7 g ) was added , followed by fmoc - osu ( 1 . 39 g ). the reaction mixture was stirred at room temperature for 16 hours . water ( 50 ml ) was added . the acetone was removed under reduced pressure . the aqueous was washed with diethyl ether ( 50 ml ), acidified with 1n hcl to ph 3 , extracted with methylene chloride ( 3 × 150 ml ). the extracts were washed with dilute brine ( 50 ml ), dried over mgso 4 , concentrated to give a foamy white solid . the crude white solid was purified by column chromatography with methanol / ethyl acetate ( 3 / 7 , v / v ) as eluent to give the clean product as a white solid ( 1 . 3 g ) in 86 % yield . reaction 5 illustrates the synthesis of a β - amino acid wherein the exocyclic amino substituent the nitrogen heteroatom is in the 4 - position relative to the ring nitrogen . compound 52 : compound 51 ( 2 . 0 g ) and nabh 3 cn ( 0 . 54 g ) were dissolved in methanol ( 40 ml ), 1n hcl ( aqueous ) was added dropwise to maintain ph 3 - 4 . after 15 - 20 minutes , ph change slowed . the mixture was stirred for an additional 1 . 0 hour , while 1n hcl was added occasionally to keep ph 3 - 4 . water ( 100 ml ) was added . the mixture was extracted diethyl ether ( 3 × 150 ml ). the extracts were washed with 1n nahco 3 ( 100 ml ) and dilute brine ( 100 ml ), dried over mgso 4 , and concentrated to give a colorless oil ( 1 . 9 g ) in 95 % yield . the product was used directly without further purification . compound 53 : compound 52 ( 1 . 9 g ) and ph 3 p ( 2 . 8 g ) were dissolved in toluene ( anhydrous , 30 ml ) under nitrogen . a solution of diethyl azodicarboxylate ( 1 . 5 ml ) in toluene ( 10 ml ) was subsequently introduced via syringe over 15 minutes . the reaction mixture was stirred under nitrogen at room temperature for 12 hours . the toluene was removed under reduced pressure . the residue was purified by column chromatography with ethyl acetate / hexane ( 3 / 7 , v / v ) as eluent to afford a colorless oil ( 1 . 6 g ) in 91 % yield . compound 54 : compound 53 ( 1 . 0 g ) and r -(+)- α - methylbenzylamine ( 1 . 1 ml ) were mixed with water ( 15 ml ). the mixture was stirred at 55 ° c . for 67 hours . the mixture was taken up in diethyl ether ( 300 ml ), and the aqueous layer was separated . the ether solution was washed with water ( 3 × 50 ml ), dried over mgso 4 , and concentrated to give a slight yellow oil . the diastereometric isomers were separated by column chromatography with ethyl acetate / hexane ( 2 / 8 , v / v ) as eluent to give rss ( 0 . 2 g ) and rrr ( 0 . 34 g ) in 51 % overall yield . compound 55 : compound 54 ( 4 . 2 g ) was dissolved in ethyl acetate ( 200 ml ). 4n hcl in dioxane ( 4 . 35 ml ) was added dropwise while stirring . a white precipitate resulted . the ethyl acetate was removed under reduced pressure , and the resulting white solid ( 4 . 6 g , 100 %) was dried in vacuo . compound 56 : compound 55 ( 4 . 6 g ) was dissolved in 95 % ethanol ( 150 ml ) in a hydrogenation flask . 10 % palladium on activated carbon ( 0 . 5 g ) was added . the flask was pressurized with hydrogen to 50 psi , rocked at room temperature for 22 hours , by which time nmr spectroscopy indicated that the hydrogenolysis was complete . the pd / c was removed by filtration . the filtrate was concentrated to give a white solid . the white solid was dissolved in acetone / water ( 2 / 1 , v / v , 150 ml ). nahco 3 ( 9 . 7 g ) was added , followed by cbz - osu ( 3 . 4 g ). the reaction mixture was stirred at room temperature for 14 hours . water ( 100 ml ) was added . the acetone was removed under reduced pressure . the aqueous mixture was extracted with ethyl acetate ( 3 × 200 ml ). the extracts were washed with 1n hcl ( 3 × 100 ml ) and saturated nahco 3 ( aqueous ), dried over mgso 4 , and concentrated to give a colorless oil . the crude product was purified by column chromatography with ethyl acetate / hexane ( 3 / 7 , v / v ) as eluent to give the clean product as a colorless sticky oil ( 4 . 0 g ) in 90 % yield . compound 57 : compound 56 ( 2 . 0 g ) was dissolved in methanol / water ( 3 / 1 , v / v , 115 ml ), cooled to 0 ° c ., lioh . h 2 o ( 2 . 4 g ) was added . the mixture was stirred at 0 ° c . for 15 hours , by which time tlc indicated that the hydrolysis was complete . saturated ammonium hydroxide ( aqueous , 100 ml ) was added . the methanol was removed under reduced pressure . the aqueous was acidified with 1n hcl to ph 3 , extracted with ethyl acetate ( 3 × 200 ml ). the extracts were washed with dilute brine ( 100 ml ), dried over mgso 4 , concentrated to give a foamy solid ( 1 . 63 g , 88 %), which was used directly without further purification ). compound 58 : compound 57 ( 1 . 63 g ) was dissolved in methanol ( 70 ml ) in a hydrogenation flask . 5 % palladium on activated carbon ( 250 mg ) was added . the flask was pressurized with hydrogen to 35 psi , rocked at room temperature for 15 hours , by which time nmr spectroscopy indicated that the hydrogenolysis was complete . the pd / c was removed by filtration . the filtrate was concentrated to give a white solid . the white solid was dissolved in acetone / water ( 2 / 1 , v / v , 90 ml ), cooled to 0 ° c . nahco 3 ( 2 . 27 g ) was added , followed by fmoc - osu ( 1 . 83 g ). the reaction mixture was stirred at 0 ° c . for 2 hours , then at room temperature for 28 hours . water ( 50 ml ) was added . the acetone was removed under reduced pressure . the aqueous was acidified with 1n hcl to ph 3 , extracted with ethyl acetate ( 3 × 200 ml ). the extracts were washed with dilute brine ( 100 ml ), dried over mgso 4 , concentrated to give a foamy white solid . the crude white solid was purified by column chromatography with methanol / ethyl acetate ( 3 / 7 , v / v ) as eluent to give the clean product as a white solid ( 1 . 68 g ) in 84 % yield . solution - phase synthesis of a β - peptide chain containing alternating residues of unsubstituted cyclohexane rings and amino - substituted cyclohexane rings proceeds in conventional fashion as outlined in reaction 6 : reaction 6 works with equal success to build peptides wherein the residues are the same or different . reaction 7 is an illustration of a homologation reaction combined with conventional solution - phase peptide synthesis , to yield a β - peptide having acyclic - substituted residues alternating with cyclically - constrained residues : as noted above , the β - peptides of the present invention can be substituted with any number of substituents , including hydroxy , linear or branched c 1 - c 6 - alkyl , alkenyl , alkynyl ; hydroxy - c 1 - c 6 - alkyl , amino - c 1 - c 6 - alkyl , c 1 - c 6 - alkyloxy , c 1 - c 6 - alkyloxy - c 1 - c 6 - alkyl , amino , mono - or di - c 1 - c 6 - alkylamino , carboxamido , carboxamido - c 1 - c 6 - alkyl , sulfonamido , sulfonamido - c 1 - c 6 - alkyl , urea , cyano , fluoro , thio , c 1 - c 6 - alkylthio , mono - or bicyclic aryl , mono - or bicyclic heteraryl having up to 5 heteroatoms selected from n , o , and s ; mono - or bicyclic aryl - c 1 - c 6 - alkyl , heteroaryl - c 1 - c 6 - alkyl , and combinations thereof . effecting such substitutions is well within the set of skills possessed by a synthetic peptide chemist . for example , appending a sulfonamido moiety to the cylic backbone substituent can be accomplished in conventional fashion using reaction 8 . compound 63 : compound 61 ( 90 mg ) was dissolved in 4 nhcl in dioxane ( 2 . 0 ml ). the reaction mixture was stirred for 1 . 5 hours . the dioxane was then removed in vacuo . the residue was dissolved in pyridine ( 2 . 0 ml ), then cooled to 0 ° c . in an ice - bath . methanesulfonylchloride ( 71 μi ) was added dropwise . after the addition , the reaction mixture was stirred at room temperature for 12 hours . the pyridine was then removed in vacuo . the residue was taken up in ethyl acetate ( 50 ml ). the mixture was washed with dilute brine ( 2 × 10 ml ), dried over mgso 4 , and concentrated to give the clean product as a colorless oil ( 70 mg ) in 82 % yield . compound 64 : compound 62 ( 30 mg ) was dissolved in 4 n hcl in dioxane ( 2 . 0 ml ). the reaction mixture was stirred for 1 . 5 hours . the dioxane was then removed in vacuo . the residue was dissolved in pyridine ( 1 . 0 ml ), then cooled to 0 ° c . in an ice - bath . toluenesulfonylchloride ( 63 mg ) was added in portions . after the addition , the reaction mixture was stirred at room temperature for 12 tours . the pyridine was then removed in vacuo . the residue was taken up in methylene chloride / dithyl ether ( 1 / 1 , v / v , 100 ml ). the mixture was washed with dilute brine ( 3 × 20 ml ), dried over mgso 4 , and concentrated to give a liquid residue . the crude product was purified by column chromatography with ethyl acetate / hexane ( 4 / 6 , v / v ) as eluent to give the clean product as a colorless oil ( 25 g ) in 74 % yield . analogous reactions will append a carboxyamido group . cyclic imino carboxylic acids : cyclic imino carboxylic acids are a class of β - amino acid residues wherein the amino functionality itself is part of a ring structure . these β - amino acids can also be used as residues in the present invention . general experimental procedure a . peptide couplings using bop - cl as the coupling reagent . boc - xxx - obn ( 1 . 0 eq .) was dissolved in 4 n hcl / dioxane ( 2 . 5 eq .). the solution was stirred for 2 h , the solvent was removed under a stream of n 2 , and the residue was dried under vacuum to give a white solid ( xxx - obn - hcl ). this material was dissolved in methylene chloride ( 0 . 1 m ). boc - xxx - oh ( 1 . 0 eq .) was added and the reaction mixture was cooled to 0 c . bopcl ( 1 . 0 eq .) was added , followed by diea ( 2 . 0 eq .). the reaction mixture was stirred for 48 h at 5 c in the cold room . the reaction mixture was removed from the cold room and poured into a solution of diethyl ether ( 3 × reaction volume ) and h 2 o ( 2 × reaction volume ). the organic layer was isolated and washed with saturated khso4 , saturated nahco 3 , and brine . the organic layer was dried over mgso 4 and concentrated . the crude product was then purified by column chromatography to give boc -( xxx ) 2 - obn . general experimental procedure b . peptide couplings using bop - cl as the coupling reagent . boc - xxx - obn ( 1 . 0 eq .) was dissolved in 4 n hcl / dioxane ( 2 . 5 eq .). the solution was stirred for 2 h , the solvent was removed under a stream of n 2 , and the residue was dried under vacuum to give a white solid ( xxx - obn - hcl ). this material was dissolved in methylene chloride ( 0 . 2 m ). boc - xxx - oh ( 1 . 0 eq .) was added and the reaction mixture was cooled to 0 c . bopcl ( 1 . 0 eq .) was added , followed by diea ( 2 . 5 eq .). the reaction mixture was stirred for 48 h at room temperature . the reaction mixture was poured into a solution of diethyl ether ( 3 × reaction volume ) and h 2 o ( 2 × reaction volume ). the organic layer was isolated and washed with saturated khso4 , saturated nahco 3 , and brine . the organic layer was dried over mgso 4 and concentrated . the crude product was then purified by column chromatography to give boc -( xxx ) 2 - obn . boc -( s )- nip - obn or boc -( r )- nip - obn is the building block for the synthesis of nipecotic acid oligomers . the protected monomer was synthesized in three steps beginning with a resolution via co - crystallization with (+) or (−) csa . the amino group was then protected as the tert - butyl carbamate , and the carboxyl group was protected as the benzyl ester . ( s )- nip -( s )- csa 1 . 1 . ( 1s )-(+)- 10 - camphorsulfonic acid ( 11 . 62 g , 0 . 05 mol ) was added to a stirred solution of racemic nipecotic acid ( 6 . 46 g , 0 . 05 mol ) in acetone ( 100 ml ). the solution was heated to reflux , and h 2 o ( 15 ml ) was added until all solids dissolved . the solution was cooled to room temperature and allowed to stir overnight . the precipitate that formed was isolated by filtration and recrystallized three times with acetone / h 2 o ( 6 / 1 , v / v ) to afford 1 . 99 g ( 11 % yield ) of the desired product as a white solid : m . p . 221 - 223 ° c . ; { a } d + 25 . 30 ( c 1 . 0 , meoh ). boc -( s )- nip - oh 1 . 2 . ( s )- nip -( s )- csa ( 1 . 90 g , 5 . 3 mmol ) was dissolved in methanol ( 12 ml ). triethylamine ( 2 . 2 ml , 15 . 8 mmol ) and di - tert - butyl dicarbonate ( 1 . 38 g , 6 . 3 mmol ) were added and the solution was stirred at 50 c for 12 h . the solution was then concentrated , and the residue was dissolved in h 2 o . the aqueous solution was washed with diethyl ether , and the organic layer was discarded . the aqueous layer was acidified to ph 3 with 1 m hcl and extracted with ch 2 cl 2 . the organic layer was dried over mgso 4 and concentrated to afford 1 . 24 g ( quantitative yield ) of the desired product as a white solid : m . p . 166 - 168 ° c . ; fab - ms m / z ( m + na + ) calcd for c11h19no4na 252 . 3 , obsd 252 . 5 . boc -( s )- nip - obn 1 . 3 . boc -( s )- nip - oh ( 0 . 70 g , 3 . 1 mmol ) was dissolved in n , n - dimethylformamide ( dmf ) ( 14 ml ). cs 2 co 3 ( 1 . 0 g , 3 . 1 mmol ) and benzyl bromide ( 0 . 41 ml , 3 . 5 mmol ) were added , and the solution was stirred at room temperature for 24 h . the solution was then concentrated , and the residue was dissolved in h 2 o . the aqueous solution was then extracted with ch 2 cl 2 . the organic solution was dried over mgso 4 and concentrated to give an oil . the crude product was purified by column chromatography eluting with ethyl acetate / hexanes ( 1 / 3 , v / v ) to afford 0 . 81 g ( 82 % yield ) of the desired product as a white solid . oligomers of nipecotic acid were synthesized in a stepwise fashion using standard coupling procedures : boc -( s )- nip - n ( me ) 2 1 . 4 . via general procedure a , hcl - n ( me ) 2 ( 0 . 29 g , 3 . 5 mmol ) was coupled with boc -( s )- nip - oh ( 0 . 4 g , 1 . 7 mmol ). after workup , the crude product was purified by column chromatography eluting with ethyl acetate / hexanes ( 1 / 1 , v / v ) to afford 0 . 29 g ( 65 % yield ) of the desired product as a colorless oil ; fab - ms m / z ( m + na + ) calcd for c 11 h 19 no 4 na + 279 . 3 , obsd 279 . 1 . boc -{( s )- nip } 2 - obn 1 . 5 . via general procedure a , boc ( s )- nip - obn ( 0 . 80 g , 2 . 5 mmol ) was boc - deprotected and coupled with boc -( s )- nip - oh ( 0 . 64 g , 2 . 5 mmol ). after workup , the crude product was purified by column chromatography eluting with ethyl acetate / hexanes ( 1 / 1 , v / v ) to afford 0 . 69 g ( 64 % yield ) of the desired product as a colorless oil ; maldi - tof - ms m / z ( m + na + ) calcd for c 24 h 34 n 2 o 5 na + 453 . 5 , obsd 453 . 3 . boc -{( s )- nip } 3 - obn 1 . 6 . via general procedure a , boc {( s )- nip -} 2obn ( 0 . 37 g , 0 . 85 mmol ) was boc - deprotected and coupled with boc -( s )- nip - oh ( 0 . 19 g , 0 . 85 mmol ). after workup , the crude product was purified by column chromatography eluting with ethyl acetate / methanol ( 10 / 1 , v / v ) to afford 0 . 22 g ( 49 % yield ) of the desired product as a white foam ; maldi - tof - ms m / z ( m + na + ) calcd for c 30 h 43 n 3 o 6 na + 564 . 3 , obsd 564 . 3 . boc -{( s )- nip } 4 - obn 1 . 7 . via general procedure a , boc {( s )- nip -} 2 obn ( 0 . 29 g , 0 . 62 mmol ) was boc - deprotected and coupled with boc -{( s )- nip } 2 - oh ( 0 . 29 g , 0 . 85 mmol ). after workup , the crude product was purified by column chromatography eluting with ethyl acetate / methanol ( 10 / 1 , v / v ) to afford 0 . 27 g ( 68 % yield ) of the desired product as a white foam ; maldi - tof - ms m / z ( m + na + ) calcd for c 36 h 52 n 4 o 7 na + 675 . 4 , obsd 675 . 4 . boc -{( s )- nip } 5 - obn 1 . 8 . via general procedure a , boc -( s )- nip - obn ( 88 . 4 mg , 0 . 28 mmol ) was boc - deprotected and coupled with boc -{( s )- nip } 4 - oh ( 0 . 14 g , 0 . 28 mmol ). after workup , the crude product was purified by column chromatography eluting with ethyl acetate / methanol ( 10 / 1 , v / v ) to afford 0 . 11 g ( 52 % yield ) of the desired product as a white foam ; maldi - tof - ms m / z ( m + na + ) calcd for c 42 h 61 n 5 o 8 na + 786 . 4 , obsd 786 . 5 . boc -{( s )- nip } 6 - obn 1 . 9 . via general procedure a , boc {( s )- nip -} 2 obn ( 0 . 46 g , 1 . 1 mmol ) was boc - deprotected and coupled with boc -{( s )- nip } 4 - oh ( 0 . 16 g , 0 . 3 mmol ). after workup , the crude product was purified by column chromatography eluting with ethyl acetate / methanol ( 10 / 1 , v / v ) to afford 0 . 12 g ( 45 % yield ) of the desired product as a white foam ; maldi - tof - ms m / z ( m + n + ) calcd for c 48 h 70 n 6 o 9 na + 897 . 5 , obsd 897 . 6 . circular dichroism data for 0 . 5 mm nip pentamer in isopropanol as a function of temperature indicate that the nip oligomers are thermally stable , and that only at 75 ° c . does the stability of the oligomer decrease ( data not shown ). circular dichroism data for 0 . 5 mm nip hexamer ( 25 ° c .) protected in methanol and deprotected in h 2 o , ph = 7 . 6 suggest that the same secondary structure is adopted in h 2 o , with there being a small decrease in the stability of the structure ( data not shown ). the synthesis of this monomer is an extension of that given in klein et al . ( 1997 ), bio . & amp ; med . chem . let . 7 : 1773 . 3 - hydroxy -( r )- pyrrolidine . trans - 4 - hydroxy - l - proline ( 13 . 11 g , 0 . 1 mol ) was added to cyclohexanol ( 65 ml ), followed by the addition of 2 - cyclohexene - 1 - one ( 0 . 65 ml ). the reaction mixture was heated at 180 ° c . until all solids were dissolved . the solution was cooled to room temperature and concentrated by vacuum rotary evaporation . the crude product was carried on to the next synthetic step without further purification . 3 - hydroxy - cbz -( r )- pyrrolidine 2 . 1 . 3 - hydroxy -( r )- pyrrolidine ( 8 . 71 g , 0 . 1 mol ) was dissolved in ch 2 cl 2 ( 260 ml ) and cooled to 0 ° c . triethylamine ( 33 . 5 ml , 0 . 24 mol ) and benzyl chloroformate ( 14 . 9 ml , 0 . 11 mol ) were added , and the resulting solution was stirred for 2 h at 0 ° c . the solution was gradually warmed to room temperature and allowed to stir overnight . the solution was washed with 1 m hcl , saturated nahco 3 , and brine . the organic solution was dried over mgso 4 and concentrated . the crude product was purified by column chromatography eluting with ethyl acetate to afford 13 . 7 g ( 62 % yield , 2 steps ) of the desired product as a purple oil . 3 - tosyl - cbz -( r )- pyrrolidine 2 . 2 . 3 - hydroxy - cbz -( r )- pyrrolidine ( 13 . 7 g , 0 . 06 mol ) was dissolved in ch 2 cl 2 ( 250 ml ) and cooled to 0 ° c . p - toluenesulfonyl chloride ( 14 . 16 g , 0 . 07 mol ), and triethylamine ( 20 . 7 ml , 0 . 15 mol ) were added and the resulting solution was stirred for 4 h at 0 ° c . the solution was washed with 1 m hcl , saturated nahco 3 , and brine . the organic solution was dried over mgso 4 and concentrated . the crude product was purified by column chromatography eluting with ethyl acetate / hexanes ( 3 / 1 , v / v ) to afford 20 . 4 g ( 88 % yield ) of the desired product as an oil . 3 - cyano - cbz -( s )- pyrrolidine 2 . 3 . 3 - tosyl - cbz -( r )- pyrrolidine ( 20 . 4 g , 0 . 05 mol ) was dissolved in dmso ( 54 ml ), followed by the addition of kcn ( 5 . 3 g , 0 . 08 mol ). the reaction mixture was stirred for 5 h at 80 ° c . the solution was cooled to room temperature and brine / h 2 o ( 90 ml ) ( 1 / 1 , v / v ) was added . the aqueous solution was extracted with ethyl acetate . the organic extracts were dried over mgso 4 , and concentrated . the crude product was purified by column chromatography eluting with ethyl acetate / hexanes ( 1 / 1 , v / v ) to afford 8 . 13 g ( 65 % yield ) of the desired product as an oil . cbz -( s )- pca - ome 2 . 4 . 3 - cyano - cbz -( s )- pyrrolidine ( 8 . 13 g , 35 . 3 mmol ) was dissolved in methanol ( 35 ml ), followed by the addition of concentrated hcl ( 35 ml ). the solution was stirred for 3 days at room temperature . the solution was neutralized by nahco 3 . the methanol was removed and the solution was diluted with h 2 o ( 100 ml ). the aqueous solution was extracted with ch 2 cl 2 . the organic extracts were dried over mgso 4 and concentrated . the crude product was purified by column chromatography eluting with ethyl acetate / hexanes ( 1 / 1 , v / v ) to afford 5 . 26 g ( 57 % yield ) of the desired product as a colorless oil . boc -( s )- pca - ome 2 . 5 . cbz -( s )- pca - ome ( 5 . 26 g , 20 . 0 mmol ) was dissolved in methanol ( 0 . 1 m ), 10 % pd / c ( 12 % vol ), and boc 2 o ( 5 . 67 g , 25 . 9 mmol ) were added , and the solution was shaken on a parr appartus for 12 h under pressurized h 2 . the solution was filtered through a plug of glass wool , and the filtrate was concentrated . the crude product was purified by column chromatography eluting with ethyl acetate / hexanes ( 1 / 1 , v / v ) to afford 3 . 79 g ( 83 % yield ) of the desired product as an colorless oil . boc -( s )- pca - oh 2 . 6 . boc -( s )- pca - ome ( 2 . 52 g , 11 . 0 mmol ) was dissolved in methanol ( 155 ml ) and h 2 o ( 54 ml ) and the solution was cooled to 0 ° c . lioh . h 2 o ( 4 . 6 g , 0 . 11 mol ) was added , followed by h 2 o 2 ( 6 . 23 ml , 0 . 05 mol ) and the solution was stirred for 15 h in the cold room at 5 ° c . while still cold , na 2 so 3 ( 21 g , 0 . 17 mol ) in h 2 o ( 93 ml ) was added . the methanol was removed and the solution was brought to ph 2 with 1 m hcl . the aqueous solution was extracted with methylene chloride . the organic extracts were dried over mgso 4 and concentrated to afford 2 . 36 g ( 88 % yield ) of the desired product as a white solid . boc -( s )- pca - obn 2 . 7 . boc -( s )- pca - oh ( 1 . 07 g , 4 . 9 mmol ) was dissolved in dmf ( 50 ml ). cs 2 co 3 ( 1 . 62 g , 4 . 9 mmol ) and benzyl bromide ( 0 . 63 ml , 5 . 2 mmol ) were added , and the solution was stirred for 24 h at room temperature . the solution was then concentrated , and the residue was dissolved in h 2 o . the aqueous solution was then extracted with ethyl acetate . the organic solution was dried over mgso 4 and concentrated . the crude product was purified by column chromatography eluting with ethyl acetate / hexanes ( 1 / 3 , v / v ) to afford 1 . 52 g ( 90 % yield ) of the desired product as a white solid . boc -( s )- pca - n ( me ) 2 2 . 8 . via general procedure a , hcl - n ( me ) 2 ( 31 . 7 mg , 0 . 5 mmol ) was coupled with boc -( s )- pca - oh ( 93 . 5 mg , 0 . 4 mmol ). after workup , the crude product was purified by column chromatography eluting with ethyl acetate / hexanes ( 1 / 1 , v / v ) to afford 39 . 1 mg ( 37 % yield ) of the desired product as a colorless oil . boc -{( s )- pca } 2 - obn 2 . 9 . via general procedure a , boc -( s )- pca - obn ( 0 . 46 g , 1 . 5 mmol ) was boc - deprotected and coupled with boc -( s )- pca - oh ( 0 . 32 g , 1 . 5 mmol ). after workup , the crude product was purified by column chromatography eluting with ethyl acetate / hexanes ( 3 / 1 , v / v ) to afford 0 . 28 g ( 47 % yield ) of the desired product as a colorless oil . boc -{( s )- pca } 3 - obn 2 . 10 . via general procedure a , boc -{( s )- pca } 2 - obn ( 90 mg , 0 . 2 mmol ) was boc - deprotected and coupled with boc -( s )- pca - oh ( 48 mg , 0 . 2 mmol ). after workup , the crude product was purified by column chromatography eluting with ethyl acetate / methanol ( 20 / 1 , v / v ) to afford 29 . 8 mg ( 27 % yield ) of the desired product as an colorless oil . boc -{( s )- pca } 4 - obn 2 . 11 . via general procedure a , boc -{( s )- pca } 2 - obn ( 0 . 21 g , 0 . 5 mmol ) was boc - deprotected and coupled with boc -{( s )- pca } 2 - oh ( 0 . 16 g , 0 . 5 mmol ). after workup , the crude product was purified by column chromatography eluting with ethyl acetate / methanol ( 20 / 1 , v / v ) to afford 67 . 9 mg ( 27 % yield ) of the desired product as a white foam ; maldi - tof - ms m / z ( m +) calcd for c 32 h 44 n 4 o 7 na 620 . 724 , obsd 620 . 5 . boc -{( s )- pca } 5 - obn 2 . 12 . via general procedure a , boc -{( s )- pca } 3 - obn ( 0 . 10 g , 0 . 2 mmol ) was boc - deprotected and coupled with boc -( s )- pca - oh ( 36 mg , 0 . 2 mmol ). after workup , the crude product was purified by column chromatography eluting with methylene chloride / methanol ( 20 / 1 , v / v ) to afford 16 . 3 mg ( 14 % yield ) of the desired product as a clear , glassy solid ; maldi - tof - ms m / z ( m +) calcd for c 37 h 51 n 5 o 8 na 716 . 841 , obsd 716 . 5 . boc -{( s )- pca } 6 - obn 2 . 13 . via general procedure a , boc -{( s )- pca } 4 - obn ( 0 . 10 g , 0 . 2 mmol ) was boc - deprotected and coupled with boc -{( s )- pca } 2 - oh ( 52 mg , 0 . 2 mmol ). after workup , the crude product was purified by column chromatography eluting with methylene chloride / methanol ( 20 / 1 , v / v ) to afford 7 . 0 mg ( 6 % yield ) of the desired product as a clear , glassy solid ; maldi - tof - ms m / z ( m +) calcd for c 42 h 58 n 6 o 9 na 813 . 957 , obsd 813 . 5 . the synthesis of this monomer is an extension of that given in patel et al . ( 1997 ), j . org . chem . 62 : 6439 : n - tert - butoxycarbonyl - l - serine - β - lactone 3 . 1 . a solution of triphenylphosphine ( 7 . 48 g , 28 . 5 mmol ) in anhydrous thf ( 110 ml ) was stirred under n 2 , cooled to 78 ° c ., and dimethylazodicarboxylate ( 4 . 83 ml , 30 . 7 mmol ) was added dropwise . the mixture was stirred for 10 min , and a solution of boc - serine ( 4 . 5 g , 21 . 9 mmol ) in thf ( 110 ml ) was added dropwise . after the addition , stirring was continued at 78 ° c . for 30 min , and for an additional 3 h after the cooling bath had been removed . the solution was concentrated , and the residue was purified by column chromatography eluting with hexanes / ethyl acetate ( 2 / 1 , v / v ) to afford 2 . 21 g ( 54 % yield ) of the desired product as a white solid . ( s )- n2 -( tert - butoxycarbonyl )- n3 -( 2 - propenyl )- 2 , 3 - diaminopropanoic acid 3 . 2 . a n - tert - butoxycarbonyl - l - serine - β - lactone ( 2 . 21 g , 11 . 8 mmol ) in acetonitrile ( 224 ml ) was added dropwise to a stirred solution of allylamine ( 21 . 9 ml , 0 . 29 mmol ) in acetonitrile ( 448 ml ). the solution was stirred for 2 h at room temperature and then concentrated . the solid residue was slurried with acetonitrile and filtered to afford 1 . 51 g ( 52 % yield ) of the desired product as a white solid . ( s )- n2 -( tert - butoxycarbonyl )- n3 -( benzyloxycarbonyl )- n3 -( 2 - propenyl )- 2 , 3 - diaminopropanoic acid 3 . 3 . a solution of ( s )- n2 -( tert - butoxycarbonyl )- n3 -( 2 - propenyl )- 2 , 3 - diaminopropanoic acid ( 2 . 80 g , 11 . 4 mmol ) in saturated nahco 3 ( 36 ml ) and h 2 o ( 5 ml ) was treated dropwise with a solution of benzyl chloroformate ( 1 . 84 ml , 12 . 8 mmol ) in acetone ( 2 . 5 ml ). the cloudy reaction mixture was stirred for 2 h . the resulting solution was partitioned between diethyl ether ( 130 ml ) and h 2 o ( 65 ml ). the aqueous layer was cooled in an ice bath , brought to ph 2 with 1 m hcl , and extracted with ethyl acetate . the organic extracts were dried over mgso 4 and concentrated to afford 3 . 15 g ( 73 % yield ) of the desired product as a colorless oil . ( s )- n1 -( tert - butoxycarbonyl )- n4 -( benzyloxycarbonyl )- piperazine carboxylic acid 3 . 5 . a solution of ( s )- n2 -( tert - butoxycarbonyl )- n3 -( benzyloxycarbonyl )- n3 -( 2 - propenyl )- 2 , 3 - diaminopropanoic acid ( 3 . 15 g , 8 . 3 mmol ) in methylene chloride ( 110 ml ) and methanol ( 11 ml ) was cooled to − 78 ° c . under n 2 . ozone was passed through the solution until a pale blue color persisted ( 6 psi o 2 , 90 v , 20 min ). the excess ozone was purged by bubbling n 2 through the solution for 15 min . dimethyl sulfide ( 11 ml ) was added , and the solution was allowed to warm gradually to room temperature overnight . after 20 h , the reaction mixture was diluted with methylene chloride ( 200 ml ) and washed with brine . the organic layer was dried over mgso 4 and concentrated to afford 3 . 02 g ( 95 % yield ) of the desired product as a yellow foam . the crude material and triethylsilane ( 1 . 4 ml , 8 . 8 mmol ) in methylene chloride ( 200 ml ) under n 2 were cooled to − 78 ° c . and treated dropwise with boron trifluoride diethyl etherate ( 1 . 11 ml , 8 . 8 mmol ). after 30 min , more triethylsilane ( 1 . 4 ml , 8 . 8 mmol ) and boron trifluoride diethyl etherate ( 1 . 11 ml , 8 . 8 mmol ) were added in a similar fashion . the reaction mixture was stirred for 2 h at − 78 ° c ., brine was added , and the cold mixture was extracted with methylene chloride . the organic extracts were dried over mgso 4 and concentrated . the crude product was purified by column chromatography eluting with methylene chloride / ethyl acetate / acetic acid ( 2 / 1 / 0 . 03 , v / v / v ) to afford 2 . 13 g ( 74 % yield ) of the desired product as a white solid . ( s )- n1 -( tert - butoxycarbonyl )- n4 -( benzyloxycarbonyl )- piperazine ethyl ester 3 . 6 . ( s )- n1 -( tert - butoxycarbonyl )- n4 -( benzyloxycarbonyl )- piperazine carboxylic acid ( 4 . 66 g , 12 . 8 mmol ) was dissolved in dmf ( 128 ml ). cs 2 co 3 ( 4 . 37 g , 13 . 4 mmol ) and ethyl iodide ( 1 . 23 ml , 15 . 3 mmol ) were added and the reaction mixture was stirred for 24 h at room temperature . the reaction mixture was concentrated , and the residue was dissolved in h 2 o . the aqueous solution was extracted with ethyl acetate . the organic layer was dried over mgso 4 and concentrated . the crude product was purified by column chromatography eluting with ethyl acetate / hexanes ( 1 / 1 , v / v ) to afford 3 . 77 g ( 75 % yield ) of the desired product as an oil . ( s )- n1 -( mesyl )- n4 -( benzyloxycarbonyl )- piperazine ethyl ester 3 . 7 . ( s )- n1 -( tert - butoxycarbonyl )- n4 -( benzyloxycarbonyl )- piperazine ethyl ester ( 3 . 77 g , 9 . 6 mmol ) was dissolved in 4 n hcl / dioxane and stirred for 2 h at room temperature . the reaction mixture was concentrated under a stream of n2 , then on the vacuum line . the residue was dissolved in methylene chloride and cooled to 0 ° c . triethylamine ( 6 . 7 ml , 50 mmol ) and dmap ( 0 . 12 g , 1 . 0 mmol ) were added , followed by methanesulfonyl chloride ( 1 . 5 ml , 19 . 2 mmol ). the reaction solution was stirred for 24 h at room temperature . the reaction solution was then washed with brine , and the organic layer was dried over mgso 4 and concentrated . the crude product was purified by column chromatography eluting with ethyl acetate / hexanes ( 1 / 1 , v / v ) to afford 2 . 1 g ( 59 % yield ) of the desired product as an oil . boc -{( n4 - ts )- pica } 2 - obn 3 . 8 . via general procedure a , boc -{( n4 - ts )- pica }- obn ( 0 . 15 g , 0 . 3 mmol ) was boc - deprotected and coupled with boc -{( n4 - ts )- pica }- oh ( 0 . 12 g , 0 . 3 mmol ). after workup , the crude product was purified by column chromatography eluting with ethyl acetate / hexanes ( 1 / 1 , v / v ) to afford 0 . 13 g ( 57 % yield ) of the desired product as a colorless oil . boc -{( n4 - ts )- pica } 3 - obn 3 . 9 . via general procedure a , boc -{( n4 - ts )- pica } 2 - obn ( 0 . 11 g , 0 . 2 mmol ) was boc - deprotected and coupled with boc -{( n4 - ts )- pica }- oh ( 58 . 3 mg , 0 . 2 mmol ). after workup , the crude product was purified by column chromatography eluting with ethyl acetate / hexanes ( 1 / 1 , v / v ) to afford 78 mg ( 51 % yield ) of the desired product as a white foam . boc -{( n4 - ts )- pica } 4 - obn 3 . 10 . via general procedure a , boc -{( n4 - ts )- pica } 3 - obn ( 65 . 2 mg , 0 . 1 mmol ) was boc - deprotected and coupled with boc -{( n4 - ts )- pica }- oh ( 24 . 9 mg , 0 . 1 mmol ). after workup , the crude product was purified by column chromatography eluting with ethyl acetate / hexanes ( 1 / 1 , v / v ) to afford 25 mg ( 33 % yield ) of the desired product as a white foam . circular dichroism data for ( n4 - ts )- pica oligomers in methanol ( 25 ° c .) suggest that the tetramer adopts a distinct secondary structure , which is different from the structure adopted by the dimer and trimer . trans - 4 - hydroxy - cbz - l - proline4 . 1 . benzyl chloroformate ( 8 . 6 ml , 0 . 06 mol ) was dissolved in acetone ( 12 ml ), and this solution was added dropwise to a stirred solution of trans - 4 - hydroxy - l - proline ( 6 . 56 g , 0 . 05 mol ) in satd . nahco 3 ( 160 ml ) and h 2 o ( 24 ml ). the resulting solution was stirred for 6 h at room temperature . the solution was washed with diethyl ether , and the organic layer was discarded . the aqueous layer was acidified with to ph 3 with 1 m hcl and extracted with ethyl acetate . the organic layer was dried over mgso 4 and concentrated to afford 13 . 5 g ( quantitative yield ) of the desired product as an oil . trans - 4 - tbdmso - cbz - l - proline 4 . 2 . trans - 4 - hydroxy - cbz - l - proline ( 13 . 5 g , 0 . 05 mol ) was dissolved in dmf ( 190 ml ), followed by the addition of imidazole ( 17 . 0 g , 0 . 25 mol ) and tbdms - cl ( 22 . 6 g , 0 . 15 mol ). the resulting solution was stirred for 12 h at room temperature . methanol ( 150 ml ) was added and the solution was stirred for 2 h . the solution was concentrated , the residue was dissolved in ethyl acetate and washed with 1 m hcl . the organic layer was dried over mgso 4 and concentrated . the crude product was purified by column chromatography eluting with methylene chloride / ethyl acetate / acetic acid ( 2 / 1 / 0 . 03 , v / v / v ) to afford 18 . 01 g ( 95 % yield ) of the desired product as an oil . trans - 5 - hydroxylmethyl - 3 - tbdmso - cbz - pyrrolidine 4 . 3 . trans - 4 - tbdmso - cbz - l - proline ( 14 . 31 g , 0 . 04 mol ) was dissolved in thf and added via cannula to a stirred solution of 2 m me2s . bh3 in thf ( 48 . 0 ml , 0 . 09 mol ). the resulting solution was stirred for 16 h at reflux . the reaction was then quenched with methanol ( 50 ml ) and concentrated . the residue was dissolved in ethyl acetate and washed with brine . the organic layer was dried over mgso 4 and concentrated . the crude product was purified by column chromatography eluting with hexanes / ethyl acetate ( 3 / 1 , v / v ) to afford 9 . 73 g ( 70 % yield ) of the desired product as an oil . trans - 5 - methoxymethyl - 3 - tbdmso - cbz - pyrrolidine 4 . 4 . trans - 2 - hydroxylmethyl - 4 - tbdmso - cbz - pyrrolidine ( 5 . 02 g , 13 . 7 mmol ) was dissolved in acetonitrile ( 13 . 7 ml ), followed by the addition of iodomethane ( 8 . 55 ml , 0 . 14 mol ) and ag 2 o ( 6 . 36 g , 27 . 5 mmol ). the resulting reaction mixture was stirred for 12 h at reflux in the dark . the reaction mixture was then filtered through celite and the celite was washed with acetonitrile . the filtrate was concentrated . the crude product was purified by column chromatography eluting with hexanes / ethyl acetate ( 3 / 1 , v / v ) to afford 4 . 05 g ( 78 % yield ) of the desired product as an oil . trans - 5 - methoxymethyl - 3 - tosyl - cbz - pyrrolidine 4 . 5 . trans - 2 - methoxymethyl - 4 - tbdmso - cbz - pyrrolidine ( 8 . 99 g , 23 . 6 mmol ) was dissolved in tuf ( 237 ml ), followed by the addition of 1 m tbaf in thf ( 23 . 7 ml , 23 . 7 mmol ). the resulting solution was stirred for 3 h at room temperature . the reaction was quenched with satd . nh 4 cl . the solution was concentrated , the residue dissolved in ethyl acetate and washed with brine . the organic layer was dried over mgso 4 and concentrated . the residue was dissolved in methylene chloride ( 230 ml ) and cooled to 0 ° c . dmap ( 3 . 37 g , 27 . 6 mmol ) and triethylamine ( 7 . 7 ml , 66 . 2 mmol ) were added , followed by p - toluenesulfonyl chloride ( 5 . 26 g , 27 . 6 mmol ). the reaction solution was stirred for 12 h at room temperature . the solution was washed with brine and the organic layer was dried over mgso 4 and concentrated . the crude product was purified by column chromatography eluting with hexanes / ethyl acetate ( 2 / 1 , v / v ) to afford 8 . 67 g ( 90 % yield ) of the desired product as an oil . cis - 5 - methoxymethyl - 3 - cyano - cbz - pyrrolidine 4 . 6 . trans - 5 - methoxymethyl - 3 - tosyl - cbz - pyrrolidine ( 3 . 55 g , 8 . 8 mmol ) was dissolved in dmso ( 8 . 8 ml ). finely ground nacn ( 0 . 65 g , 13 . 2 mmol ) was added , and the resulting reaction mixture was stirred 4 h at 80 ° c . the solution was cooled to room temperature , diluted with h 2 o ( 9 ml ) and brine ( 9 ml ), and extracted with ethyl acetate . the organic extracts were dried over mgso 4 and concentrated . the crude product was purified by column chromatography eluting with hexanes / ethyl acetate ( 2 / 1 , v / v ) to afford 2 . 09 g ( 87 % yield ) of the desired product as an oil . cis - 5 - methoxymethyl - boc - 3 - pyrrolidine carboxylic acid { cis - boc -( 5 - mom )- pca - oh } 4 . 7 . cis - 5 - methoxymethyl - 3 - cyano - cbz - pyrrolidine ( 1 . 71 g , 6 . 2 mmol ) was dissolved in concentrated hcl and stirred for 12 h at 50 ° c . the solution was cooled to room temperature and neutralized with nahco 3 . the solution was concentrated , and the residue was dissolved in methanol ( 62 ml ). triethylamine ( 2 . 6 ml , 18 . 7 mmol ) and boc 2 o ( 1 . 63 g , 7 . 5 mmol ) were added , and the solution was stirred 12 h at 50 ° c . the solution was concentrated and the residue was dissolved in h 2 o . the aqueous solution was washed with diethyl ether , and the organic layer was discarded . the aqueous layer was acidified with to ph 3 with 1 m hcl , and extracted with ethyl acetate . the organic layer was dried over mgso 4 and concentrated to afford 1 . 40 g ( 86 % yield ) of the desired product as an oil . cis - 5 - methoxymethyl - boc - 3 - pyrrolidine benzyl ester acid { cis - boc -( 5 - mom )- pca - obn } 4 . 8 . cis - 5 - methoxymethyl - boc - 3 - pyrrolidine carboxylic acid ( 1 . 4 g , 5 . 3 mmol ) was dissolved in dmf ,( 26 . 5 ml ). cs 2 co 3 ( 1 . 73 g , 5 . 3 mmol ) and benzyl bromide ( 0 . 76 ml , 6 . 4 mmol ) were added , and the reaction mixture was stirred 24 h at room temperature . the reaction mixture was concentrated , and the residue was dissolved in h 2 o . the aqueous solution was extracted with ethyl acetate . the organic layer was dried over mgso 4 and concentrated . the crude product was purified by column chromatography eluting with ethyl acetate / hexanes ( 1 / 1 , v / v ) to afford 1 . 88 g ( 85 % yield ) of the desired product as an oil . boc -{( cis - 5 - mom )- pca } 2 - obn 4 . 9 . via general procedure b , cis - boc -( 5 - mom )- pca - obn ( 1 . 88 g , 5 . 38 mmol ) was boc - deprotected and coupled with cis - boc -( 5 - mom )- pca - oh ( 1 . 40 g , 5 . 38 mmol ). after workup , the crude product was purified by column chromatography eluting with ethyl acetate / hexanes ( 3 / 1 , v / v ) to afford 1 . 90 g ( 72 % yield ) of the desired product as an oil . boc -{( cis - 5 - mom )- pca } 3 - obn 4 . 10 . via general procedure b , cis - boc -{( 5 - mom )- pca } 2 - obn ( 0 . 26 g , 0 . 54 mmol ) was boc - deprotected and coupled with cis - boc -( 5 - mom )- pca - oh ( 0 . 13 g , 0 . 54 mmol ). after workup , the crude product was purified by column chromatography eluting with ethyl acetate / methanol ( 20 / 1 , v / v ) to afford 0 . 25 g ( 75 % yield ) of the desired product as a white foam . boc -{( cis - 5 - mom )- pca } 4 - obn 4 . 11 . via general procedure b , cis - boc -{( 5 - mom )- pca } 2 - obn ( 0 . 26 g , 0 . 54 mmol ) was boc - deprotected and coupled with cis - boc -{( 5 - mom )- pca } 2 - oh ( 0 . 20 g , 0 . 54 mmol ). after workup , the crude product was purified by column chromatography eluting with ethyl acetate / methanol ( 15 / 1 , v / v ) to afford 0 . 24 g ( 57 % yield ) of the desired product as a white foam . boc -{( cis - 5 - mom )- pca } 5 - obn 4 . 12 . via general procedure b , cis - boc -{( 5 - mom )- pca } 3 - obn ( 0 . 12 g , 0 . 18 mmol ) was boc - deprotected and coupled with cis - boc -{( 5 - mom )- pca } 2 - oh ( 0 . 09 g , 0 . 20 mmol ). after workup , the crude product was purified by column chromatography eluting with methylene chloride / methanol ( 10 / 1 , v / v ) to afford 0 . 12 g ( 83 % yield ) of the desired product as a white foam . boc -{( cis - 5 - mom )- pca } 6 - obn 4 . 13 . via general procedure b , cis - boc -{( 5 - mom )- pca } 4 - obn ( 0 . 13 g , 0 . 17 mmol ) was boc - deprotected and coupled with cis - boc -{( 5 - mom )- pca } 2 - oh ( 0 . 07 g , 0 . 17 mmol ). after workup , the crude product was purified by column chromatography eluting with methylene chloride / methanol ( 10 / 1 , v / v ) to afford 70 mg ( 52 % yield ) of the desired product as a glassy solid . boc -( cis - 5 - mom )- pca - nme 2 4 . 14 . via general procedure b , boc - cis -( 5 - mom )- pca - obn ( 0 . 11 g , 0 . 41 mmol ) was boc - deprotected and coupled with dimethylamine hydrochloride ( 0 . 04 g , 0 . 49 mmol ). after workup , the crude product was purified by column chromatography eluting with ethyl acetate / hexanes ( 3 / 1 , v / v ) to afford 57 mg ( 50 % yield ) of the desired product as an oil . the following monomers have also been synthesized via a similar reaction scheme : compound 1 . trans - 4 - hydroxy - l - proline ( 13 . 11 g , 0 . 1 mol ) was dissolved in 1m naoh ( 120 ml ) and cooled to 0 ° c . boc 2 o ( 24 . 0 g , 0 . 11 mol ) dissolved in dioxane ( 120 ml ) was added and the solution was stirred for 4 h at room temperature . dioxane was removed via rotary evaporation and the resulting aqueous solution was washed with ether . the aqueous solution was then brought to ph 2 with 1 m hcl and extracted with ethyl acetate . the organic extracts were dried over mgso 4 and concentrated to afford 21 . 3 g ( 92 % yield ) of the desired product as a white foam . boc - trans - 4 - hydroxy - l - proline ( 9 . 27 g , 0 . 04 mol ) was dissolved in a 1m benzene / methanol ( 5 / 1 , v / v ) solution . 2m tmschn 2 / hexanes was added dropwise and the resulting solution was stirred at room temperature for 1 h . the reaction solution was concentrated via rotary evaporation . the crude product was carried on to the next synthetic step without further purification . boc - trans - 4 - hydroxy - l - proline methyl ester ( 10 . 35 g , 0 . 04 mol ) was dissolved in dmf ( 135 ml ). imidazole ( 13 . 6 g , 0 . 20 mol ) was added , followed by the addition of tert - butyldimethylsilyl chloride ( 18 . 1 g , 0 . 12 mol ) and the solution was stirred overnight at room temperature . dmf was removed via vacuum rotary evaporation and the residue was dissolved in ethyl acetate . the solution was washed with 1m hcl and brine . the organic solution was dried over mgso 4 and concentrated . the crude product was purified by column chromatography eluting with hexanes / ethyl acetate ( 4 / 1 , v / v ) to afford 12 . 83 g ( 89 % yield ) of the desired product 1 as a colorless oil . compound 2 . diisopropyl amine ( 16 . 2 ml , 0 . 12 mol ) was dissolved in thf ( 215 ml ) and cooled to 0 ° c . 2 . sm n - butyllithium / hexanes was added and the solution was stirred for 30 minutes at 0 ° c . the solution was then cooled to 20 ° c . and hmpa ( 60 ml , 0 . 34 mol ) was added . after 10 minutes , a solution of 1 ( 27 . 4 g , 0 . 076 mol ) in thf ( 215 ml ) was added dropwise . after addition the reaction solution was stirred from 20 ° c . to 0 ° c . the solution was then cooled to 78 ° c . and methyl iodide ( 23 . 7 ml , 0 . 38 mol ) was added . the reaction solution was stirred for 2 h at 78 ° c . and then quenched with saturated nh 4 cl . the resulting solution was extracted with ethyl acetate . the organic extracts were dried over mgso 4 and concentrated . the crude product was purified by column chromatography eluting with hexanes / ethyl acetate ( 6 / 1 , v / v ) to afford 21 . 09 g ( 74 % yield ) of the desired product 2 as a colorless oil . see ohtake et al . ( 1999 ), bull . chem . soc . japan 72 : 2737 . compounds 3 . nabh 4 ( 6 . 14 g , 0 . 16 mol ) was added to a stirred solution of 2 ( 24 . 26 g , 0 . 065 mol ) in thf ( 150 ml ). the resulting solution was stirred at reflux for 4 h . meoh ( 50 ml ) was added over 1 h , and the solution was then diluted with h 2 o . the organic solvents were removed via rotary evaporation and the resulting aqueous solution was extracted with ethyl acetate . the organic extracts were dried over mgso 4 and concentrated . the crude product was purified by column chromatography eluting with hexanes / ethyl acetate ( 3 / 1 , v / v ) to afford 17 . 76 g ( 83 % yield ) of the desired product 3 as a colorless oil . compound 4 . 3 ( 15 . 19 g , 0 . 046 mol ) was dissolved in ch 3 cn . methyl iodide ( 28 . 7 ml , 0 . 46 mol ) was added , followed by the addition of ag 2 o ( 25 g , 0 . 092 mol ). the reaction mixture was stirred overnight at 55 ° c ., and then filtered through celite . the resulting filtrate was concentrated and purified by column chromatography eluting with hexanes / ethyl acetate ( 4 / 1 , v / v ) to afford 13 . 88 g ( 88 % yield ) of the desired product 4 as a colorless oil . compound 5 . 1m tbaf / thf ( 42 ml , 0 . 042 mol ) was added dropwise to a solution of 4 ( 13 . 88 g , 0 . 04 mol ) in thf ( 200 ml ). the solution was stirred for 3 h at room temperature and the quenched with saturated nh 4 cl . the organic solvent was removed via rotary evaporation , and the resulting aqueous solution was extracted with ethyl acetate . the organic extracts were dried over mgso 4 and concentrated . the crude product was carried on to the next synthetic step without further purification . the crude product was dissolved in ch 2 cl 2 ( 200 ml ) and cooled to 0 ° c . dmap ( 5 . 9 g , 0 . 049 mol ) and triethylamine ( 13 . 5 ml , 0 . 099 mol ) were added , followed by p - toluenesulfonyl chloride ( 8 . 8 g , 0 . 049 mol ), and the resulting solution was stirred overnight at room temperature . the solution was washed with 1m hcl , and brine . the organic solution was dried over mgso 4 and concentrated . the crude product was purified by column chromatography eluting with hexanes / ethyl acetate ( 4 / 1 , v / v ) to afford 13 . 76 g ( 85 % yield ) of the desired product 5 as a colorless oil . compound 6 . 5 ( 13 . 76 g , 0 . 034 mol ) was dissolved in dmso ( 17 ml ). 18 - crown - 6 ( 5 eq ) and finely ground kcn ( 5 eq ) were added and the reaction mixture was stirred for 6 h at 80 ° c . the reaction solution was diluted with h 2 o ( 17 ml ) and then extracted with ethyl acetate . the organic solution was dried over mgso 4 and concentrated . the crude product was purified by column chromatography eluting with hexanes / ethyl acetate ( 4 / 1 , v / v ) to afford 13 . 76 g ( 69 % yield ) of the desired product 6 as a colorless oil . compound 7 . 6 ( 4 . 34 g , 0 . 017 mol ) was dissolved in concentrated hcl ( 68 ml ) and stirred 6 h at 50 ° c . the solution was diluted with h 2 o , neutralized with nahco 3 , and concentrated . the crude product was carried on to the next synthetic step without further purification . triethylamine ( 9 . 5 ml , 0 . 068 mol ) and boc 2 o ( 7 . 5 g , 0 . 034 mol ) were added to a stirred solution of the resulting crude product in meoh ( 170 ml ). the reaction mixture was stirred overnight at reflux . the reaction mixture was diluted with h 2 o , the meoh was removed via rotary evaporation , and the resulting aqueous solution was washed with ether . the aqueous solution was then brought to ph 2 with 1 m hcl and extracted with ethyl acetate . the organic extracts were dried over mgso 4 and concentrated to afford 3 . 55 g ( 76 % yield ) of 7 as the desired product as a white foam . compound 8 . 7 ( 2 . 3 g , 8 . 4 mmol ) was dissolved in a 1m benzene / methanol ( 5 / 1 , v / v ) solution . 2m tmschn 2 / hexanes ( 4 . 4 ml , 8 . 8 mmol ) was added dropwise and the resulting solution was stirred at room temperature for 1 h . the reaction solution was concentrated by rotary evaporation . the crude product was purified by column chromatography eluting with hexanes / ethyl acetate ( 1 / 1 , v / v ) to afford 2 . 4 g ( quantitative yield ) of the desired product 8 as a colorless oil . a . general procedure for synthesis of free amine . boc -( disub pca ) n - ome was dissolved in 4n hcl / dioxane ( 2 . 5 eq .) the solution was stirred for 2 h , the solvent was removed under a stream of n 2 , and the residue was dried under vacuum to give the desired product , hcl . h -( disub pca ) n - ome , as a white solid . b . general procedure for synthesis of boc protected amine . boc -( disub pca ) m - oh and hcl . h -( disub pca ) n - ome were dissolved in ch 2 cl 2 ( 0 . 1 m ) and cooled to 0 ° c . bopcl ( 2 . 0 eq .) was added , followed by diea ( 5 eq .) the reaction mixture was stirred for 48 h at room temperature . the solution was washed with 1m hcl , saturated nahco 3 , and brine . the organic solution was dried over mgso 4 and concentrated . the crude product was purified by column chromatography to give the desired product , boc -( disub pca ) n + m - ome . c . general procedure for synthesis of isobutyl amide . boc -( disub pca ) n + m - ome was dissolved in 4n hcl / dioxane ( 2 . 5 eq .) the solution was stirred for 2 h , the solvent was removed under a stream of n 2 , and the residue was dried under vacuum to give the desired product , hcl . h -( disub pca ) n + m - ome , as a white solid . this residue was dissolved in ch 2 cl 2 and cooled to 0 ° c . iso - cl ( 2 eq .) was added , followed by diea ( 5 eq .) the reaction solution was stirred for 24 h at room temperature . the solution was washed with 1m hcl , saturated nahco 3 , and brine . the organic solution was dried over mgso 4 and concentrated . the crude product was purified by column chromatography to give the desired product , iso -( disub pca ) n + m - ome . β - peptide 101 is constructed from β - substituted β - amino acid residues (“ β 3 - residues ”), which were made in enantiomerically pure form from the corresponding α - amino acid residues using a methodology developed by seebach et al . 15 β - peptides like 101 can be conveniently prepared with conventional automated solid - phase peptide synthesis equipment . both the tat fragment 102 and β - peptide analog 101 bear n - terminal fluorescein labels , to allow detection of cell membrane transit via fluorescence microscopy . hela cells were used to measure cell membrane transit . hela cells were obtained from the american type culture collection ( 10801 university boulevard , manassas , va . 20110 - 2209 ). hela cells were cultured in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ) supplemented with 10 % ( v / v ) fetal bovine serum ( fbs ), penicillin ( 100 units / ml ), and streptomycin ( 100 μg / ml ) at 37 ° c . in a humidified incubator containing 5 % co 2 . all studies were performed using asynchronous log - phase cultures . exponentially growing hela cells were dissociated with trypsin , plated 40 % confluent on six - well lab - tek coverslips ( nunc inc ., naperville , ill .) and cultured overnight . the culture medium was discarded , and the cells were washed once with dmem . the peptides and fluorescein stock solutions were diluted in dmem , and the cell monolayers were incubated at 37 ° c . with peptide solutions at the appropriate concentrations for 10 min . subsequently , cells were rinsed three times with dmem at room temperature and fixed in paraformaldehyde ( 4 % w / v in pbs ) for 5 min at room temperature . for experiments at 4 ° c ., the protocol was the same except that all incubations were performed at 4 ° c . until the end of the fixation procedure . fixed cell monolayers were permeabilized with “ triton x - 100 ”- brand detergent ( 0 . 1 % w / v ) for 10 min and washed twice with pbs ( ph 7 . 3 ). cells were stained with propidium iodide ( 1 μg / ml in pbs ) for 5 min at room temperature . cells were then washed three times with pbs and mounted onto glass microscope slides using mounting medium for fluorescence . the distribution of the fluorescence was analyzed on a zeiss axiovert 100tv confocal microscope equipped with a kr / ar laser and 63 × oil immersion objective lens ( leica ). images were captured with biorad laser sharp mrc1024 and adobe photoshop version 4 . 0 . 1 software . β - peptide 101 ′ is the same fragment as shown in 101 , with the exception that the n - terminal fluorescein group is no longer present , nor is the c - terminal amino group . in 101 ′, x is hydrogen , an amino - capping group , an amino - protecting group , or a molecule or compound of interest ; y is hydrogen , a carboxy - capping group , a carboxy - protecting group , or a molecule or compound of interest . as used herein , the term “ protecting group ” denotes generically any chemical moiety capable of selective addition to and removal from a reactive site to allow manipulation of a chemical entity at sites other than the protected site ( s ). the term “ capping group ” denotes generically any chemical moiety capable of selective addition to a reactive site , but not necessarily selectively removable from the site once bound ( e . g ., acetyl , formyl , or anisyl when used as an n - terminal capping group ). a host of capping and / or protecting groups are known in the art . an illustrative , non - limiting list of protecting groups includes methyl , formyl , ethyl , acetyl , t - butyl , anisyl , benzyl , trifluoroacetyl , n - hydroxysuccinimide , t - butoxycarbonyl , benzoyl , 4 - methylbenzyl , thioanisyl , thiocresyl , benzyloxymethyl , 4 - nitrophenyl , benzyloxycarbonyl , 2 - nitrobenzoyl , 2 - nitrophenylsulphenyl , 4 - toluenesulphonyl , pentafluorophenyl , diphenylmethyl , 2 - chlorobenzyloxycarbonyl , 2 , 4 , 5 - trichlorophenyl , 2 - bromobenzyloxycarbonyl , 9 - fluorenylmethyloxycarbonyl , triphenylmethyl , and 2 , 2 , 5 , 7 , 8 - pentamethyl - chroman - 6 - sulphonyl . the substituents x and y , may be the same or different , and may or may not be orthogonally removable . in the present invention , compound 101 ′ and related analogs , homologs , and peptides containing compound 101 ′ as a portion of a larger polypetide ( containing either α - amino acids , β - amino acids , or a combination thereof ) are to be used as the “ vehicle ” portion of a conjugate to translocate a molecule across the membrane of a living cell . thus , in the preferred embodiment of the present invention , a molecule of interest ( i . e ., a molecule that is to be translocated across the cell membrane ) is conjugated to a vehicle comprising 101 ′ and derivative thereof . the resulting conjugate is then contacted with the living cell . the presence of the vehicle causes the entire conjugate to pass through the cell membrane and into the interior of the target cell . as used herein , the term “ contacted ,” explicitly encompasses bringing the β - peptide or β - peptide conjugate into physical contact with the living cell by any means , without limitation , including ( by way of illustration only ), direct contact in vitro or in vivo , or bringing the β - peptide or β - peptide conjugate into physical contact with the living cell via any route of administration to a higher living organism ( including mammals ), including orally , topically , nasally , via inhalation , parenterally , intravenously , intra - arterially , intramuscularly , and rectally . the nature of the molecule or compound attached to the β - peptide vehicle is not critical to the invention , so long as the entire conjugate will translocate across a living cell membrane . it is very much preferred that the molecule be conjugated to the vehicle at the vehicle &# 39 ; s n - terminus . the molecule may be attached at an interior position . the molecule may also be attached at the c - terminus of the vehicle , but this point of attachment is not preferred . the molecule conjugated to the β - peptide vehicle need not have pharmacological activity . for example , the molecule conjugated to the β - peptide vehicle can be a dye , a fluorophore , a chromophore , a radio - label , a contrast agent , etc . as shown in the following paragraphs , such conjugates are useful for visualizing the interior of cells after exposure to the conjugates . fig1 a through 1e show representative fluorescence results with hela cells for 101 and 102 . neither molecule moves significantly into the cells when introduced at a concentration of 10 nm or 50 nm ( fig1 a and 1 b ), but at 100 nm , 500 nm , and 1000 nm , both compounds 101 and 102 can be detected inside the cells ( fig1 c , 1 d and 1 e ). to identify the major intracellular destination of these molecules , the cells were co - stained with propidium iodide , which is selective for nucleic acids . the co - staining results with tat fragment 102 ( see fig2 ) match previous reports that related peptides localize in the nucleus ; the results for β - peptide 101 show that this unnatural analog behaves similarly to its a - peptide counterpart . control experiments were also conducted to determine whether all 11 β - amino acid residues were necessary for translocation by 101 and 102 . working from tat 48 - 60 , vivès et al . showed that arg - 55 , arg - 56 and arg - 57 were critical for translocation ( in contrast , the presence or absence of pro - 58 , pro - 59 and / or gln - 60 had little effect on translocation ). 2b therefore , the truncated β - peptide 103 , which lacks the three c - terminal β 3 - homoarginine residues of 101 , was examined for translocation , as was the corresponding α - peptide analog 104 ( see fig3 ). no cell penetration could be detected after treatment with up to 1 μm 103 or 104 , a ten - fold higher concentration than is required for detectable cell penetration by full length β - peptide 101 and its α - analog 102 . in addition , fluorescein itself did not enter the cells under these conditions . these results show that a minimum β - peptide length and / or charge is required for movement across cell membranes . to investigate the ability of the disclosed compounds to form helical structures , circular dichroism spectra were obtained for β - peptide 101 ′ ( see fig4 ). these results indicate that β - peptide 101 ′ displays the characteristic 14 - helical cd signature , with a minimum at 216 and a maximum at 199 nm . thus , β - peptide 101 ′ has an intrinsically higher propensity than does α - peptide 102 to adopt an ordered conformation in non - aqueous environments . the mechanism by which short tat - derived peptides and related peptides traverse cell membranes remains unclear . several groups have concluded that endocytosis does not play a role in this process because the transit rate is not temperature - dependent . 2a the inventors found no difference in translocation activity for either 101 or 102 between 37 ° c . and 4 ° c . these findings are consistent with prior reports with regard to α - peptide 102 and suggest that cell penetration by β - peptide 101 does not depend upon endocytosis . other β - peptide conjugate fabricated according to the above protocols have also be found to translocate across cell membranes , including β - peptides wherein the backbone of the molecule is rigidified by cyclically - constrained β - amino acid residues . these are residues wherein the α and β carbons of the backbone are incorporated into a ring structure . thus , the following compounds have also been synthesized and shown to translocate across the cell membrane of living hela cells : based on these results , it is also expected that the following compounds will likewise translocate across the cell membranes of living cells : the invention is not restricted to those embodiments illustrated above , but encompasses all modifications and variations thereof as fall within the scope of the attached claims . 1 . prochiantz , a . curr . opin . cell biol . 2000 , 12 , 400 . schwarze , s . r . ; hruska , k . a . ; dowdy , s . f . trends cell biol . 2000 , 10 , 290 . lingren , m . ; hällbrink , m . ; prochiantz , a . ; langel , ü . trends pharmacol . sci . 2000 , 21 , 99 . 2 . 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