the synthetic modification 
 of proteins enables the construction 
 of biomolecular hybrids that can be used to study protein function , deliver potent therapeutics to cellular targets , and build new materials . the synthesis of these constructs requires a suite of chemoselective 
 bioconjugation reactions that proceed under mild , aqueous conditions 
 in the presence of the native functional groups that are present on 
 protein surfaces . the most common methods for protein modification 
 target the nucleophilic side - chains of lysine and cysteine . however , these strategies can 
 result in complex product mixtures , as lysine is typically found in 
 high abundance on the protein surface and 
 uniquely reactive cysteine labeling sites can be difficult to install 
 in many instances ( such as thiol proteases and proteins produced via 
 the eukaryotic secretory pathway , for example ) . many newer approaches 
 for the site - selective modification of proteins 
 involve the introduction of artificial amino acids with reactivities 
 that are orthogonal to those of the native 
 amino acids . along these lines , a number of powerful methods have 
 been developed for the selective modification of azide , alkyne , alkene , carbonyl , and aniline moieties . however , the difficulty 
 of introducing a non - canonical amino acid can limit the application 
 of these methods . complementary approaches rely on the site - selective 
 modification of native amino acids by enzymes . in addition , a reliable method for the modification of c - terminal 
 thioesters with n - terminal cysteines , termed  native chemical 
 ligation  , has been developed by kent and co - workers . this method has been used for the semi- and total synthesis of complex 
 protein substrates , including the chemical synthesis of a single glycoform 
 of human erythropoietin . as an alternative 
 strategy , we and others have developed methods 
 for the selective modification of the n - terminal amino group . methods that target the n - terminus can offer significant advantages 
 for bioconjugate preparation , as they can be used for a wide range 
 of protein targets produced by virtually any expression system . conceptually 
 powerful as they are , however , these methods can be hampered by long 
 reaction times , often require large excesses of reagent , and/or involve 
 at least two - steps for the attachment of synthetic molecules . we have 
 therefore sought to develop new techniques that can achieve n - terminal 
 modification with similarly high positional selectivity , but with 
 significantly improved efficiency . herein , we report an oxidative 
 coupling pathway that can preferentially 
 modify the n - terminus of proteins with fast kinetics . peptide substrates 
 were first used to screen reaction conditions and identify the site 
 of modification . a peptide panel with varying n - terminal residues was then evaluated to determine the sequence specificity of the reaction , 
 leading to the identification of proline as the optimal n - terminal 
 amino acid . the reaction was next applied to protein substrates , showing 
 similarly high levels of conversion when an n - terminal proline residue 
 was present . this mild bioconjugation reaction enables the facile , 
 rapid modification of proteins to create a well - defined and stable 
 linkage in a single position , and thus should be useful for many different 
 applications in chemical biology and the construction of biomolecular 
 materials . we have previously reported 
 the chemoselective coupling of aniline 
 moieties on proteins to electron - rich aromatic rings , such as o - aminophenols , at slightly acidic ph ( 6.06.5 ) . these reactions require the addition of naio4 or k3fe(cn)6 as a terminal oxidant , with the latter 
 reagent exhibiting improved compatibility with glycoproteins and substrates 
 with free sulfhydryl groups . the use of ferricyanide as the oxidant 
 also yields a single reaction product ( 1 ) , whereas periodate 
 leads to the formation of a ring contracted species as a competing 
 pathway . the ferricyanide - based reactions 
 are presumed to involve an o - iminoquinone as the 
 reactive intermediate , as suggested in scheme 1 , or could involve the corresponding o - quinone after 
 imine hydrolysis . taken together , the oxidative coupling strategies 
 have demonstrated excellent functional group compatibility and the 
 ability to join large unprotected biomolecules at low concentrations , 
 as demonstrated for the coupling of peptides , polymers , and nucleic 
 acids to specific locations on viral capsids and antibody fc domains . while these coupling reactions 
 were found to be highly aniline - selective 
 under the conditions used , several studies have reported the reaction 
 of o - aminophenols and o - catechols 
 with native amino acids , dating back to 1949 . in addition , recent work by messersmith has shown the ability of 
 proteins to be coupled to o - quinone moieties present 
 on polydopamine - coated surfaces . these reports 
 suggested that secondary coupling pathways could be developed to achieve 
 the modification of native amino acids with o - aminophenols 
 ( scheme 1 ) , and thus initial experiments were 
 designed to identify the optimal reaction conditions for achieving 
 this with complex molecules . in our previous 
 work , we noted that low amounts of background reactivity could be 
 observed in aniline - based oxidative coupling reactions when higher 
 ph conditions ( > ph 6.5 ) were used . in 
 an initial effort to characterize this alternative reaction pathway , 
 conditions and reaction times were first screened to increase the 
 reaction yields for peptides that did not contain aniline groups . angiotensin i and melittin were used as substrates , as they contain 
 many reactive amino acids , including lys , arg , his , trp , and tyr . the peptides were exposed to 2-amino - p - cresol using 
 k3fe(cn)6 as the oxidant . the reaction ph was 
 varied from 5.5 to 8.5 , and the reaction mixtures were analyzed using 
 maldi - tof ms ( supporting information figure 
 s1 ) . the level of modification increased with the basicity of the 
 reaction , with near quantitative modification of angiotensin i after 
 20 min at ph 7.5 and higher . throughout these initial investigations , 
 it was noted that angiotensin and melittin showed significant differences 
 in reactivity , with angiotensin consistently demonstrating better 
 conversion . ms / ms analysis of the angiotensin product was used to 
 identify the participating residue , and revealed that the n - terminal 
 amino group was responsible for the observed reactivity ( supporting information figure s2 ) . as further 
 confirmation of the site - selectivity , several peptide substrates were 
 screened for reactivity ( figure 1a and supporting information figure s3 ) . consistent 
 with the n - terminal reaction selectivity , the only peptide that did 
 not react had a pyroglutamate in this position , and therefore no free 
 amino group . ( a ) 
 modification of commercially available peptides was monitored by maldi - tof 
 ms . ( b ) a positional scan of the n - terminal amino acid was 
 evaluated . peptides with the sequence xadswag were tested for reactivity with 2-amino - p - cresol . the reactions were run with 100 m peptide , 200 m 
 aminophenol , and 5 mm ferricyanide at ph 7.5 and analyzed by lc  ms . shown is the average percent modification with error bars representing 
 the standard deviation of three reactions . the same peptides were 
 used for a screen of coupling partner equivalents ( c ) and a time screen 
 ( d ) . most buffers did not alter the reactivity , but 
 imidazole and buffers containing a morpholine or piperazine ring ( pipes , 
 hepes , hepps , and mops ) significantly impeded the reaction ( supporting information figure s4 ) . this is possibly 
 due to small amounts of buffer impurities that react competitively 
 with the oxidized intermediate . the time course of the reaction was 
 also investigated ( supporting information figure s5 ) . in addition , it was found that the peptides could be modified 
 using naio4 as the oxidant , or 4-methylcatechol as the 
 coupling partner ( supporting information figures s67 ) . both of these reactions showed the same dependence 
 on ph ; however , moderate levels of modification were still observed 
 at acidic ph ( 5.56.5 ) when using these alternative coupling 
 conditions . these observations suggested that the peptides were reacting 
 with the o - quinone intermediate formed in 
 situ from either the catechol or the iminoquinone precursor 
 ( after imine hydrolysis ) . given the differential 
 reactivity observed on peptide substrates , we synthesized peptides 
 with varied n - terminal residues ( xadswag ) to determine the specificity of the reaction . the base sequence 
 was selected to increase the mass of the peptide , impart water solubility , 
 and include a tryptophan residue for quantitation using uv monitoring . the peptides were synthesized on the solid phase using standard fmoc 
 synthesis , cleaved from the resin , and purified by hplc . after purification , 
 the peptides were resuspended in phosphate buffer , ph 7.5 , adjusted 
 to a concentration of 1 mm , and stored at 20 c until 
 use . to assess the effect of the n - terminal residue on reactivity , 
 the peptides ( 100 m ) were reacted with 2 equiv of 2-amino - p - cresol ( 200 m ) in the presence of k3fe(cn)6 ( 5 mm ) in phosphate buffer , ph 7.5 ( figure 1b ) . ms ( see supporting information figure s8 for representative ms data 
 for the modified peptides ) . most n - terminal amino acids showed good - to - high 
 levels of conversion ( 6090% ) , but proline stood out as the 
 only residue that showed nearly complete modification ( 90100% ) . a second observation of this screen was the fact that tryptophan , 
 tyrosine , and methionine residues were not oxidized by the ferricyanide 
 reagent , consistent with our previous report of oxidative coupling 
 with this oxidant ( see supporting information figure s8 ) . however , free cysteine residues can be oxidized to various 
 species , potentially including disulfides and sulfenic acids , and 
 thus it is recommended that they be protected as disulfides before 
 oxidative coupling is attempted ( vide infra ) . to optimize the reagent ratios ( specifically the equivalents of o - aminophenol ) , the peptides ( 100 m ) were reacted 
 with 110 equiv of the o - aminophenol ( 1001000 
 m ) in the presence of ferricyanide ( 10 mm ) . after 30 min , the 
 reactions were quenched with excess tris(2-carboxyethyl)phosphine 
 ( tcep ) . it was demonstrated that conversion was highest using 25 
 equiv of the coupling partner ( figure 1c ) . 
 using more than 5 equiv of this was most likely due to the ability of 
 the aminophenol to react with itself at higher concentrations ( 1 
 mm ) . consistent with this , when using 10 equiv of the o - aminophenol , a byproduct was observed with a mass that corresponded 
 to the condensation of 3 aminophenols ( 344 da ) . we also investigated 
 the differences in coupling rates for representative 
 n - termini . the reaction of 2-amino - p - cresol with 
 three different peptides was monitored over the course of 1 h ( figure 1d ) . the peptides ( 100 m ) were reacted with 
 2 equiv of the o - aminophenol ( 200 m ) in the 
 presence of ferricyanide ( 5 mm ) , and aliquots were quenched with excess 
 tcep at the indicated time points . the proline terminal peptide not 
 only reached the highest level of conversion , but also did so in a 
 significantly shorter time than the other termini . despite efforts 
 to optimize conditions for all n - termini the reaction of n - terminal 
 amines with o - aminophenols 
 was characterized using small molecule mimics . the methyl esters of 
 phenylalanine ( h - phe - ome ) and proline ( h - pro - ome ) were coupled to 
 2-amino - p - cresol using ferricyanide at ph 7.5 . the 
 crude products were characterized using two - dimensional nmr and high - resolution 
 mass spectrometry . the primary amine of h - phe - ome formed p - iminoquinone product 2 , which was analogous to the 
 one formed with aniline coupling partners ( scheme 1 , supporting information figure 
 s9 ) . however , the secondary amine of proline prevented the formation 
 of the p - iminoquinone tautomer , and thus favored o - quinone product 3 ( scheme 1 , supporting information figure 
 s10 ) . given the different linkage obtained with proline , we verified 
 the stability of the product to a variety of conditions . the proline 
 terminal peptide , padswag , was first modified with 2-amino - p - cresol . after purification , the modified peptide ( 100 
 m ) was exposed to reductants , nucleophiles and acidic and basic 
 ph ( 10 mm additives or buffer ) . no loss of product was 
 observed under any of the conditions tested , demonstrating the hydrolytic 
 stability of the product ( supporting information figure s11 ) . the ability of the linkage to withstand these conditions 
 renders this method quite useful for the construction of biomolecular 
 materials for a variety of applications . with a view toward in vivo applications , current efforts are examining the 
 stability of the linkage in blood plasma , as well as evaluating the 
 intrinsic immunogenicity of the o - quinone group . in the process of characterizing the reaction products , it was observed 
 that the colored products absorbed light at wavelengths greater than 
 500 nm ( with max between 505 and 525 nm depending 
 on the amine coupling partner ) . as the starting coupling partners 
 and ferricyanide did not absorb at these wavelengths , this unique 
 absorbance provided a means to monitor the reaction progress . the 
 different amine coupling partners ( p - toluidine , h - pro - ome , 
 and h - phe - ome ) were reacted with 4-methylcatechol in the presence 
 of 10 mm ferricyanide , and the absorbance of the resulting solution 
 was monitored at 520 nm to determine the relative rates of reactivity 
 ( figure 2a ; for unnormalized data see supporting information figure s12 ) . the catechol 
 substrate was used for these studies to simplify the reaction pathway 
 by eliminating the imine hydrolysis step . the reactions were run under 
 pseudo - first order conditions with 0.1 mm catechol and 1 mm amine 
 coupling partner . when the reaction was carried out at ph 6.0 , the 
 reaction with the proline analogue reached completion nearly as rapidly 
 ( 2 min ) , but the reaction with the primary aliphatic amine 
 of phenylalanine required longer reaction times ( 10 min ) . ( a ) amine coupling partners were reacted with 4-methylcatechol 
 as a model substrate . reactions were run under pseudo - first 
 order conditions with 100 m catechol , 1 mm amine , 10 mm ferricyanide 
 in 50 mm phosphate buffer . ( b ) a peptide containing both an n - terminal 
 proline and a p - aminophenylalanine residue ( pad(paf)swag ) was tested for reactivity with 2 equiv of 2-amino - p - cresol at ph 6 . the remainder of the reaction was purified 
 and then reacted with 2 equiv of the aminophenol at ph 7.5 and analyzed 
 by lc  ms . by quantifying product 
 formation by absorbance , we were also able 
 to measure the second - order rate constant for the proline - based coupling 
 ( supporting information figure s13 ) . the 
 reaction of 1 equiv of h - pro - ome ( 100 m ) with 1 equiv of 4-methylcatechol 
 ( 100 m ) and 100 equiv of k3fe(cn)6 ( 10 
 mm ) was performed in triplicate at 25 c . the second - order rate 
 constant for the coupling was determined to be 44  4 m s. while proline reacted rapidly with the electron - rich 
 coupling partner , the small molecule studies indicated that aniline 
 should react faster . the rate for the aniline reaction was too fast 
 under these conditions to determine the second - order rate constant 
 accurately . given the differences in reactivity observed 
 at ph 6.0 and 7.5 , 
 we hypothesized that it would be possible to modify the aniline side 
 chain of p - aminophenylalanine ( paf ) and the n - terminal proline amine sequentially . to test this hypothesis , 
 we synthesized a peptide containing both reactive moieties ( pad(paf)swag ) . only one modification was observed when the peptide 
 was reacted with 2-amino - p - cresol at ph 6.0 ( figure 2b ) . ms / ms analysis of the modified peptide confirmed 
 that the single modification occurred on the aniline side chain ( supporting information figure s14 ) . after this 
 step , the peptide was purified and subsequently reacted with 2-amino - p - cresol at ph 7.5 . reaction at the higher ph enabled a 
 second modification of the peptide substrate , at the n - terminal proline 
 residue . the differential reactivity 
 was investigated by comparing the reactivity of a protein containing 
 a paf residue to proteins without the artificial 
 amino acid . the paf residue was introduced into the 
 coat protein of bacteriophage ms2 , which self - assembles into a spherical , 
 hollow protein shell . myoglobin and a mutant of the tobacco mosaic 
 virus ( tmv ) coat protein were used as native protein substrates . reactions 
 with 2-amino - p - cresol were either performed on the 
 isolated , individual proteins or with the aniline containing protein 
 mixed with the native protein substrate ( supporting 
 information figure s15 ) . addition of the aniline containing 
 protein to the native protein decreased the n - terminal reactivity , 
 indicating that the aniline residues react more rapidly than n - terminal 
 residues with the o - aminophenols . in addition , ms2 
 showed significantly higher reactivity at all of the phs tested , confirming 
 preference for aniline residues . the oxidative coupling reaction 
 with n - terminal amino groups was 
 first tested on proteins with native n - termini . several proteins were 
 reacted with o - aminophenol - functionalized 5 kda peg 
 under the optimized reaction conditions ( figure 3 ) . the native proteins showed moderate levels of reactivity , which 
 could be attributed to inaccessible n - termini or simply to the less 
 reactive n - terminal residues . to test if proline terminal proteins 
 were more reactive , a proline residue was introduced to the n - terminus 
 of gfp and the tobacco mosaic virus ( tmv ) coat protein . the n - terminus 
 of tmv was also slightly extended from the native sequence ( addition 
 of pag ) . the proline - gfp was treated with a variety of conditions 
 to determine the specificity of the reaction ( figure 4a ) . only at basic ph in the presence of both the o - aminophenol substrate and the oxidant was modification observed . 
 additionally , the proline - terminal variant showed significantly improved 
 reactivity compared to that of the wild - type n - terminus . these high 
 levels of modification were maintained even when only 12 equiv 
 of the o - aminophenol peg was used . the site of modification 
 was confirmed to be the n - terminal proline by lc ms / ms analysis 
 of a tryptic digest of proline - gfp modified with 2-amino - p - cresol ( supporting information figure 
 s16 ) . ( a ) 
 the n - terminus of several proteins was pegylated using o - aminophenol - functionalized 5 kda peg and ferricyanide . ( b ) modification 
 of wild type proteins with 5 kda o - aminophenol - peg 
 was monitored by sds - page . ( a ) a proline 
 was introduced to the n - terminus of gfp . the proline terminal 
 variant showed much higher levels of modification than the wild - type 
 protein . the band doubling is due to a gel artifact , and appears in all 
 lanes . ( b ) mutants of tmv were reacted with 5 equiv of 2-amino - p - cresol and 1 mm k3fe(cn)6 for 30 min and analyzed by lc  ms . reaction conditions for both native and proline terminal 
 proteins 
 were optimized by evaluating reactivity with myoglobin and proline - gfp . the reaction time , buffer , and ph were screened ( supporting information figures s17 and s18 ) . similar to the 
 results obtained with peptide substrates , the reaction reached its 
 highest level of conversion after about 1530 min . in addition , 
 most buffer salts tested were compatible with the reaction with the 
 exception of buffers containing morpholine ( mops ) or piperazine moieties 
 ( hepes ) , as was observed with peptide substrates . these buffers decreased 
 the level of modification slightly , but did not completely inhibit 
 reactivity . the effect of reaction ph was tested using both k3fe(cn)6 and naio4 as the oxidants . little 
 reactivity was observed at acidic ph , and the level of conversion 
 increased between ph 7.0 and 8.0 . at higher reaction ph ( 8.0 ) a second modification was observed , indicating that lysines may also 
 participate in the reaction . however , it is also possible that under 
 more forcing conditions , such as higher reaction ph or increased concentration 
 of aminophenol substrate , the aminophenol reacts with both itself 
 and the n - terminal amino group resulting in double modification of 
 the n - terminus . n - terminal mutants of tmv were also evaluated 
 for their reactivity 
 with o - aminophenols . the tmv monomers assemble into 
 well - known double disk structures , displaying 34 copies of the n - terminal 
 groups on their peripheries . two n - terminal 
 mutants ( pag and ag ) were reacted with 5 equiv of 2-amino - p - cresol ( 100 m ) and 1 mm k3fe(cn)6 for 30 min . ms demonstrated that the 
 proline terminal mutant reached nearly complete conversion , while 
 the alanine terminal mutant showed low levels of modification under 
 these coupling conditions ( figure 4b , see supporting information figure s19 for wider mass 
 range and ion series ) . the compatibility of the reaction with 
 cysteine residues was also 
 tested using tmv . a single cysteine residue ( s123c ) was introduced 
 into the tmv coat protein with a proline n - terminus ( pag s123c tmv ) . this mutant was reacted with 2-amino - p - cresol and 
 analyzed by lc the cysteine residue also reacted with the o - aminophenol , resulting in two modifications . however , it was found that the n - terminal proline could be modified 
 selectively if the cysteine was first capped ( figure 5a , b , see supporting information figures s2122 for wider mass range and ion series ) . to do 
 this , the cysteine residue was protected as a disulfide bond by reaction 
 with 5,5-dithiobis(2-nitrobenzoic acid ) ( dtnb , ellman s 
 reagent ) . after the oxidative coupling step the disulfide bond was 
 readily reduced by tcep , leaving the free cysteine and the modified 
 n - terminus . alternatively , the cysteine residue was modified with 
 a maleimide , followed by modification at the n - terminus with an o - aminophenol reagent . a ) pag s123c tmv was reacted with small molecule substrates and analyzed 
 by lc cysteine residues were protected as a disulfide using 
 ellman s reagent ( dtnb ) before oxidative coupling . subsequent 
 reduction of the disulfide resulted in selective modification of the 
 n - terminus . the n - terminal 
 proline was then modified with a rhodamine - functionalized o - aminophenol . this strategy allowed for the direct , dual modification of 
 the 
 protein at both the cysteine residue and the n - terminus . two fluorophores 
 paired for frster resonance energy transfer ( fret , alexa fluor 
 488 c5-maleimide and o - aminophenol functionalized 
 rhodamine b ) were thus conjugated to tmv using this strategy to create 
 a templated array of chromophores for light harvesting applications . the free cysteine was first quantitatively labeled with an alexa 
 fluor maleimide ( supporting information figure s22 ) . the n - terminal proline was then coupled to a fluorescent o - aminophenol resulting in 50% modification of the 
 tmv monomers with both fluorphores ( figure 5b ) . complete modification of the n - terminus was not observed as tmv 
 precipitated from solution with increasing levels of modification 
 with the rhodamine dye . in current experiments , we are using this 
 dual - labeling strategy to introduce more soluble chromophores . the oxidative coupling 
 reaction was also compared to the reaction 
 of protein amines with activated esters . this acylation methodology 
 is commonly employed , and can be targeted to the n - terminus by controlling 
 the reaction ph in some cases . the reactions 
 were compared on creatine kinase , a protein with a native proline 
 n - terminus . reaction with 15 equiv of o - aminophenol 
 peg resulted in good levels of modification of creatine kinase ( 5060% ) , 
 while reaction with 15 equiv of n - hydroxysuccinimide 
 ( nhs ) peg resulted in low levels of modification ( 525% , figure 6 ) . only when a vast excess of the nhs peg was used 
 were moderate levels of modification achieved . as was the case with 
 proline - gfp , some over modification was observed under the oxidative 
 coupling conditions when using five or more equivalents of aminophenol . this could result from dimerization of the oxidized species before 
 protein coupling , but has yet to be characterized due to the low abundance 
 of this product . in any case , lowering the reaction ph slightly or 
 using fewer equivalents of the o - aminophenol substrate 
 prevented the over modification from occurring . the modification of the 
 n - terminus of creatine kinase with aminophenol 
 peg was compared to the reaction of creatine kinase with nhs peg . in this study , we have identified conditions 
 for the oxidative 
 coupling of o - aminophenols to n - terminal amino acids . proline residues work particularly well with this strategy , and are 
 therefore strongly recommended when using it . these groups can be 
 introduced readily in n - terminal positions using site - directed mutagenesis 
 and escherichia coli expression , especially 
 since the methionine residue resulting from the start codon is cleaved 
 when proline is in the second position . the fast kinetics of the reaction allow it to be successful even 
 at low reagent and substrate concentrations , and suggest that it can 
 be used for sterically demanding bioconjugations . the oxidative 
 coupling strategy reported here offers two distinct 
 advantages over other n - terminal labeling methods . first , the modification 
 occurs in a single step and does not require initial oxidation of 
 the n - terminus . second , the fast second - order kinetics allow for low 
 concentrations of the coupling partners to be used . however , to achieve 
 high levels of modification on protein substrates , proline was required 
 as the n - terminal residue . this new protein modification strategy is currently being explored 
 in our lab for the generation of protein - based materials . in the larger 
 context , new techniques for the introduction of a single functional 
 group in a specific position on a protein surface are always in demand . the ability of the n - terminal oxidative coupling method to achieve 
 this in a single , brief reaction step is highly advantageous , and 
 the fact that it can be combined with cysteine modification chemistry 
 provides new opportunities for complex bioconjugate synthesis .
the 
 synthetic modification of proteins plays an important role 
 in chemical biology and biomaterials science . 
 these fields provide 
 a constant need for chemical tools that can introduce new functionality 
 in specific locations on protein surfaces . in this work 
 , an oxidative 
 strategy is demonstrated for the efficient modification of n - terminal 
 residues on peptides and n - terminal proline residues on proteins . 
 the strategy uses o - aminophenols or o - catechols that are oxidized to active coupling species in 
 situ using potassium ferricyanide . 
 peptide screening results 
 have 
 revealed that many n - terminal amino acids can participate in this 
 reaction , and that proline residues are particularly reactive . 
 when 
 applied to protein substrates , the reaction shows a stronger requirement 
 for the proline group . 
 key advantages of the reaction include its 
 fast second - order kinetics and ability to achieve site - selective modification 
 in a single step using low concentrations of reagent . 
 although free 
 cysteines are also modified by the coupling reaction , they can be 
 protected through disulfide formation and then liberated after n - terminal 
 coupling is complete . 
 this allows access to doubly functionalized 
 bioconjugates that can be difficult to access using other methods .