The present invention relates to a method for cleaving an amide bond in a compound, thereby obtaining an N-terminal peptide as a free amine and a secondary residue , as well as an N-terminal peptide as a free amine thus obtained .

Chemical cleavage of amide (peptide ) bonds ususally require harsh conditions (March, J. Advanced organic chemistry (Wiley, New York, NY, USA, 1992 ) ; Smith, B . J . in The Protein Protocols Handbook (ed. Walker, J. M . ) 485-510 (Humana Press , Totowa, NJ, USA, 2002 ) . As a result, side reactions and the lack of specificity of chemical amide bond hydrolysis limits the scope in chemical biology and synthesis applications .

The azide functional group is exceptionally well suited for in vivo labeling of biomolecules . Azides combine a high chemical stability under biological conditions with a unique reactivity enabling mild and selective organic transformations under physiological conditions ( Saxon, E . & Bertozzi , C . R. (2000 ) Cell Surface Engineering by a Modified Staudinger Reaction . Science 287 , 2007-2010 ; Prescher, J. A. , Dube , D . H . & Bertozzi , C . R. (2004 ) Chemical remodelling of cell surfaces in living animals . Na ture 430 , 873-7 ; Kohn, M. & Breinbauer, R. (2004 ) The Staudinger Ligation - A Gift to Chemical Biology . Angew Chem Int Ed Engl 43, 3106-16) . Since azides are relatively inert to nearly all naturally occurring substances , this amino acid is truly bio-orthogonal (Van Maarseveen, J . H . & Back, J . W . (2003 ) Re-Engineering the Genetic Code : Combining Molecular Biology and Organic Chemistry . Angew Chem Int Ed Engl 42 , 5926-5928 ) , and has been used for specific modification reactions (Kiick, K. L . , Saxon, E . , Tirrell , D . A. & Bertozzi , C . R . ( 2002 ) Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation . Proc. Natl . Acad. Sci U. S. A 99, 19-24 ; Link, A. J . & Tirrell, D . A. (2003 ) Cell surface labeling of Escherichia coli via copper ( I ) - catalyzed [ 3+2 ] cycloaddition . J Am . Chem Soc. 125, 11164-11165 ) .

The amino acid Azido-homoalanine (Azhal ) has been shown to be effectively incorporated into proteins by the native methionyl tRNA synthase of E coli (Kiick et al . , supra) . Kiick et al . have demonstrated that proteins containing azidohomoalanine can be 5 selectively modified in the presence of other cellular proteins by means of Staudinger ligation with triarylphosphine reagents . It was thus suggested that incorporation of azide-functionalized amino acids into proteins in vivo provides opportunities for protein modification under native conditions and selective labeling of proteins in the 10 intracellular environment .

The present inventors now found specific hydrolysis of a peptide bond in a test octadecapeptide P ( PPHHHHHHPPRGFGXGFR, wherein X denotes Azhal ) in the presence of the phosphine TCEP, 2- mercaptoethanol or dithiothreitol . These experiments revealed a 15 product of m/z 2221 (MH ⁺ ) , due to the expected Staudinger reduction of the azide to an amine under these conditions . Surprisingly, also a second compound at m/z 1729.8 was observed in the reaction mixture . Further investigation of the unexpected cleavage product by means of low energy collision induced dissociation (CID) in an ESI-Q-FTMS mass 20 spectrometer revealed that the C-terminal portion of the peptide was '•"• intact at its N-terminus , and ran to the former position of azido- ^" S ^~f - homoalanine, which had been converted into a residue of mass 83 amu with the only possible elemental composition C ₄ H ₅ NO . This indicated that the peptide bond C-terminal to the azidohomoalanine was 25 cleaved, and that all nitrogen atoms from the azide had been lost .

After incubation of the hydrolysed peptide in IM

Tris (hydroxymethyl ) amine, two additional products were observed, at m/z 1747.8 and 1850.9 respectively, and investigation by tandem MS

30 pinpointed the modifications once again to the C-terminus . The product at 1747.8 disappeared after incubation in strong acid . This led to the conclusion that the newly formed C-terminus after cleavage was homoserine lactone (HSL) , which had been hydrolysed into homoserine (m/z 1747.8 ) , or had undergone nucleophilic attack by the

35 primary amine to yield an amide-trishydroxymethyl C terminus (m/z 1850.9 ) . The cleavage of PAN016 by TCEP in the presence of 50% ¹⁸ O enriched water showed that the amide bond adj acent C-terminal to the Azhal residue was cleaved by hydrolysis , and only one oxygen atom from water was incorporated into the N-terminal peptide .

The present inventors have thus found that upon reaction of peptide chains , e . g . proteins , containing azide-functionalized amino acids with azide-reducing agents in a protic environment cleavage of the amide bond C-terminal to azide-functionalized amino acid is accomplished to obtain a free amine compound and a secondary residue that varies depending on the azide-functionalized amino acid used . This method may advantageously be used for cleavage of proteins or peptides , but can also be used for the cleavage of other compounds comprising an amide bond C-terminal of the azide-functionalized amino acid .

Thus , the present invention relates to a method for cleaving of an amide bond in a compound comprising said amide bond and at least one azide-function spaced at 3 or 4 atoms from the carbon of the amide bond to be cleaved, said compound being chosen from the compounds of formulae I-IV :

II

, wherein Ri and R ₂ may be independently a hydrogen atom, a hydrocarbon group, or a heterocyclic group, which may have a substituent, and the two R ' s may be the same or different and independently a hydrogen atom, a hydrocarbon group, or a heterocyclic

group, which may have a substituent, said method comprising the steps of : a) providing the compound of any of formulae I-IV, wherein R ₁ and R ₂ may be independently a hydrogen atom, a hydrocarbon group, or a heterocyclic group, which may have a substituent , and the two R ' s may be the same or different and independently a hydrogen atom, a hydrocarbon group, or a heterocyclic group, which may have a substituent; and b) subj ecting the compound of step a) to an azide-reducing agent in a protic solvent to cleave the amide bond C-terminal to the non-natural amino acid, thereby obtaining RiNH ₂ and a secondary residue .

In the case of the compound to be cleaved to be a protein, it thus comprises a non-natural azide-functionalized amino acid incorporated therein, such as azidohomoalanine , azidonorvaline, and derivatives thereof ( substituted azidohomoalanine or azidonorvaline) . The non-natural azide-functionalized amino acid is located N-terminal to the amide bond to be cleaved . The skilled practitioner is aware of suitable methods for the incorporation of non-natural amino acids into compounds such as peptides and proteins . Non-limiting examples of such methods for the incorporation of non-natural amino acids into proteins or peptides include solid-phase synthesis (Marcaurelle L . A. & Bertozzi , C . R. ( 1999) New directions in the synthesis of glycopeptide mimetics . Chem . Eur. J. 5, 1384-1390 ) , native chemical ligation (Cotton, G . J. & Muir, T . W . ( 1999) Peptide ligation and its application to protein engineering . Chem . Biol . , 6 (9) , R247-256 ) , and in vitro translation protocols (Cornish, V . W . , Mendel , D . & Schultz , P . G . ( 1995 ) Probing protein structure and function with an expanded genetic code . Angew. Chem . Int . Ed. Engl . 34 , 621-633 ; Mendel , D . , Ellman, J. A. , Chang, Z . , Veenstra, D . L . , Kollman, P . A . & Schultz , P . G . ( 1992 ) Probing protein stability with unnatural amino acids . Science, 256 (5065) , 1798-1802 ) . The incorporation of azides into proteins by means of the cell ' s native translational apparatus is e . g . disclosed in Kiick et al . , supra and Link et al . , supra . All these methods are suitable for the incorporation of at least one non- natural amino acid in the peptide chain at the location to be cleaved . Detection of the presence of non-natural amino acids in a peptide chain is part of the routine work for the skilled practitioner ( see e . g . the above citations ) , and can be done by one

or more of several techniques , such as mass spectrometry, EcLman degradation, amino acid analysis, or derivatization or reaction with appropriate azido specific reagents that carry a detectable label (e . g . phosphines or alkynes , labeled with fluorophores , biotins , or any other secondary label in conj unction with a suitable assay .

The compound preferably comprises a peptide chain, and more preferably is a peptide or protein . As herein used, the term "peptide chain" encompasses any chain comprising at least two amino acids that are connected by means of an amide bond . The peptide chain may comprise any amount of amino acids of which at least two are coupled to one another by means of an amide bond, and may be prepared by in vitro or in vivo synthesis , or by chemical synthesis or native chemical ligation . Preferably, the peptide chain is a protein, more preferably a naturally occurring protein produced in an (over) expression system allowing for incorporation of non-natural amino acids , for example a system as disclosed by Kiick et al . ( supra ) , or such a protein fused to a fusion moiety, which may also be a naturally occurring protein or a chemically synthesised peptide chain .

Thus , in a preferred embodiment, R ₁ is a peptide chain (in case of the compounds of formulae III and IV) or R ₁ and R ₂ are both peptide chains ( in case of the compounds of formulae I and II ) , such that the non-natural azide-functionalized amino acid is incorporated in a peptide or a protein . R ₁ may be different from R ₂ , but R ₁ and R ₂ may also be the same .

The non-natural amino acid is preferably chosen from the group, consisting of azidohomoalanine (the azide-functionalized amino acid moiety displayed in formula I wherein both R ' s are H) , azidonorvaline (the azide-functionalized amino acid moiety displayed in formula I I wherein both R' s are H) and derivates thereof (the azide- functionalized amino acid moiety displayed either in formula I or II wherein the two R ' s may be the same or different and independently a hydrogen atom, a hydrocarbon group, or a heterocyclic group, which may have a substituent; R may e . g . be a methyl group, ethyl group, etc) , as these amino acids have the correct spacing for the reaction according to the present invention to take place . These azide amino acids display several advantages in addition to obtaining the correct

reactive group ( see figure 1 ) for cleaving of the amide bond . Azidohomoalanine , azidoalanine, azidonorvaline and azidonorleucine are shown to be methionine analogs . The azide group can survive cellular metabolism. The incorporation of the above methionine analogs into proteins is controlled most stringently by the methionyl-tRNA synthetase of the host . Thus , a methionine analog can be incorporated into the peptide chain, e . g . protein, instead of methionine itself . Using this approach, all methionines encoded in the DNA sequence are replaced by the methionine analog (see e . g . Kiick et al . , supra ) . An alternative approach is to modify an aminoacyl-tRNA such that it selectively incorporates non-natural amino acids . The latter approach is e . g . disclosed in Van Maarseveen, J . H . & Back, J . W . (2003 ) Re-engineering the genetic code : combining molecular biology and organic chemistry . Angew. Chem. Int . Ed. 42 , 5926-5928 ; Chin, J. W . , Cropp, T . A. , Anderson, J. C . Mukherj i , M . , Zhang, Z . & Schultz , P . G . (2003 ) An expanded eukaryotic genetic code . Science . 301 , 964-966; and Kwon, I . , Kirshenbaum, K. & Tirrell , D . A. (2003 ) Breaking the degeneracy of the genetic code . J. Am. Chem. Soc. 125, 7512-7513 ; Wang, L . & Schultz , P . G . ( 2005) Expanding the genetic code . Angew. Chem . Int . Ed. 44 , 34-66 ) . Using a combination of the above mentioned methods , especially those mentioned in Kwon et al . (supra) , and Chin et al .. {supra) , would allow for an in vivo overexpression system that could incorporate non-natural amino acids such as azido-functionali zed amino acids , into a ( recombinant ) protein at a position to be cleaved, and would also allow for normal methionine residues to be present at other sites at which no cleavage is desired .

In the preferred case of Ri being a peptide chain, the cleavage product RiNH ₂ will be the peptide C-terminal to the scissile bond . R _x NH ₂ will be obtained as a free amine . The secondary residue may be a lactone residue . The C-terminal end of the secondary residue is dependent on the non-natural amino acid used .

The method according to the present invention comprises as a first step a) providing the compound of any of formulae I-IV,

II

, wherein Ri and R ₂ may be independently a hydrogen atom, a hydrocarbon group, or a heterocyclic group, which may have a substituent, and the two R ' s may be the same or different and independently a hydrogen atom, a hydrocarbon group, or a heterocyclic group, which may have a substituent , as indicated above .

In step b) the compound of any of formulae I-IV is subj ected to an azide-reducing agent in a protic solvent to cleave the amide bond C-terminal to the non-natural amino acid, thereby obtaining R ₁ NH ₂ and a secondary residue .

The azide-reducing agent may be any azide-reducing agent known in the art, such as LiAlH ₄ , H ₂ /catalyst, Cr (II ) /H ⁺ , φ ₃ P/NH ₄ OH,

H ₂ S/pyridine/water, H ₂ /Lindlar catalyst, trivalent phosphines , and thiol-containing agents (Bayley H . , Standring, D . N . and Knowles , J .

R. ( 1978 ) Propane-1 , 3-dithiol : a selective reagent for the efficient reduction of alkyl and aryl azides to amines . Tetrahydr. Lett . 39, 3633-3634 ) .

A "protic solvent" as herein used, refers to a solvent that is capable of donating a hydrogen atom for hydrogen bonding . This usually requires an NH or OH bond . Non-limiting examples thereof are aqueous solutions , such as water and several types of buffer solutions , and alcohols such as ethanol , nitriles such as acetonitrile , organic acids such as acetic acid, furanes such as tetrahydrofurane , formamides such as dimethylformamide and any mixture of these solvents . The protic solvent should allow for solubilisation of the peptide chain as well as the azide-reducing agent .

As herein used, the "subj ecting the peptide chain to an azide- reducing agent in a protic solvent" refers to a reaction that occurs between the peptide chain and an azide-reducing agent, e . g . phosphine, 2-mercaptoethanol or dithiothreitol , when these are simultaneously present in the protic solvent . The peptide chain will thus be subj ected to the azide-reducing agent when a sample of peptide chain in protic solvent is mixed with the azide-reducing agent in the same protic solvent or a protic solvent that is miscible with the protic solvent used to solubilise the peptide chain .

Likely due to steric considerations , the amide bond C-terminal to the non-natural amino acid participates in the reaction with the triazene that results from the reaction of the azido moiety of this amino acid with the azide-reducing agent to accomplish cleavage of this amide bond, as is further exemplified using a phosphine as azide-reducing agent in Fig . 1. Due to the reaction with the azide- reducing agent, the resultant peptide C-terminal to the scissile bond becomes a peptide with an N-terminal free amine, whereas the resultant peptide N-terminal to the scissile bond becomes a so-called secondary residue ( Fig . 1 ) . The secondary residue is dependent on the non-natural amino acid that is used in step a) , and is e . g . a homoserine lactone residue in the case of the non-natural amino acid being azidohomoalanine . It is to be noted that homoserine and homoserine lactone are in equilibrium, and the equilibrium is dependent on pH, as the skilled practitioner is well aware of . When

the reaction is carried out at pH above 7 in the presence of a nucleophile, e . g . a primary amine, elongation of the peptide chain with the nucleophile will occur .

To the knowledge of the present inventors , this new reaction is much milder than any chemical hydrolysis of amide or peptide bonds described so far . This is also the first report describing the use of the activated aza-ylide to form free acid and amine from an amide . Until now, via aza-ylides , carboxylic acids have been turned into amides , and imidines have been formed from amides , the latter mainly in ring closure reactions . Performing this reaction in water has opened a new pathway, which may have far fetching possibilities .

It was found that the surprisingly mild cleavage reaction that can be carried out under physiological conditions without the use of harsh reactants was very effective for the cleavage of peptide chains . This is particularly advantageous for the pharmaceutical industry . Proteins may be produced in the host of choice ( see e . g . Wang & Schulz, supra) if so desired as a fusion protein as to solubilise them and may accordingly undergo regular post- translational processing, and may then be cleaved from the fusion moiety to yield the desired protein in fully processed form. The present invention finds further application in organic synthesis . It is conceivable that by use of 4-azido-butyrate or substituted derivatives thereof (in particular 2 , 2-dialkyl-4-azido butyrates ) or 5-azido valeric acid or derivatives thereof ( in particular 2 , 2- dialkyl-5-azidovalerates ) one can convert amine functions into stable amide groups , and later deprotect the amine with mild phosphine treatment as to regain the amine function .

The cleavage process proceeds in a broad range of pH values , as is further demonstrated in example 3 below . It is important that protons are available for the reaction, as is shown in fig . 1.

In a preferred embodiment, the non-natural amino acid is azidohomoalanine and the secondary residue is a homoserine lactone residue . Of the methionine analogs , azidohomoalanine is most efficiently incorporated into a protein by means of the cell ' s native translational apparatus (Kiick et al . , supra and Link et al . , supra) . Moreover, with azidohomoalanine the method according to the invention

can be very efficiently carried out as is shown below in the examples and in figure 1.

In a further preferred embodiment, the protic solvent is an aqueous solution . In such aqueous solution, protons are available for the reaction that is depicted in fig . 1.

It is preferred that the azide-reducing agent is chosen from the group, consisting of a phosphine of formula V

'P-

^R i ^R ₆ V

, wherein R ₄ , R ₅ and R ₆ are optionally substituted alkyl or aryl chains that may be the same or different, and a thiol-containing agent .

The phosphine of formula V may be any phosphine of formula V

^R 5 ^R 6

V

, wherein R ₄ , R ₅ and R ₆ are independently optionally substituted alkyl or aryl chains and may be the same or different . Of particular interest are a combination of R ₄ , R ₅ and R ₆ groups that render the phosphine soluble in a protic solvent, in particular in water . Non- limiting examples of R ₄ , R ₅ and R ₆ include carboxylic acids (e . g . propionic acid, acetic acid) , alkylamines (e . g . propylamine , ethylamine ) , alkylhydroxyls (e . g . propanols , ethanols , 2 , 3- dihydroxybutanol ) , alkylsulfonyls , or alkylguanosyls . Such phosphines are well known in the art .

The thiol-containing agent may be any thiol-containing agent known in the art, particular one of formulae VII or VIII

HS SH R ₇ SH

VIi ^vm

, wherein R ₇ is an independently optionally substituted alkyl or aryl chain . Non-limiting examples thereof include hydroxylalkylthiols , e . g . (2- ) mercaptoethanol , mercaptopropanol , mercaptobutanol , dithiothreitol , dithioerytol ; aminoalkylthiols , e . g . cysteine , cystamine ; alkylthiols , e . g . ethanedithiol , propanedithiol , butanedithiol ; and carboxyalkylthiols , e . g . thioglycolic acid, 2 , 3- dimercaptosuccinic acid. Such thiol-containing agents are readily soluble in protic solvents in the applicable pH ranges and are therefore the preferred thiol-containing agents to be used .

In the case of use of an aqueous solution as disclosed above , it is preferred that the azide-reducing agent is water-soluble such that reaction between the azide-reducing agent and the peptide chain according to the method of the invention is facilitated and most efficient .

In a further preferred embodiment of the method according to the present invention, the azide-reducing agent is a phosphine of formula V

^R 5 ^R 6

V

, wherein R ₄ , R ₅ and R ₆ are independently optionally substituted alkyl or aryl chains and may be the same or different . It was found that such phosphines are particularly suitable for use in the method of the invention .

It is preferred that the phosphine is chosen from the group, consisting of tris (carboxyethyl ) phosphine ,

tris (carboxypropyl ) phosphine , tris (hydroxyethyl ) phosphine , tris (hydroxypropyl ) phosphine , tris (ethylamine ) phosphine , tris (propylamine) phosphine . These phosphines are readily soluble in protic solvents in the applicable pH values and are therefore the preferred phosphines to be used .

It is most preferred that the phosphine is tris (carboxyethyl ) - phosphine, as this compound is readily available at a relatively low cost price . It is expected that the trisalkylaminephosphines and the trisalkylhydroxylphosphines will be particularly effective at low pH .

In an alternative embodiment of the method according to the present invention, it is preferred that the thiol-containing agent is a dithiol-containing agent, as it was found that dithiol-containing agents are 300-1 , 000 times more efficient in the reduction of the azide than monothiol-containing agents . It was found that dithiol- containing agents are about equally efficient in comparison to trivalent phosphines .

In the case of using a dithiol-containing agent , it is most preferred that dithiothreitol , butanedithiol or propanedithiol is used, as these compounds are readily available on the market, the latter two at a relatively low cost price .

It is further preferred that the compound of step a) is peptide chain, which peptide chain is obtained by in vivo incorporation of at least one non-natural azide-functionalized amino acid, since this in particular allows for post-translational processing as indicated above . The azide-functionalized amino acid may be any amino acid as disclosed above , such as azidohomoalanine and azidonorvaline and derivatives thereof .

It is also preferred that step b) of the method according to the present invention is carried out at a pH in the range of 3-10. The preferred pH is dependent on the protic solvent used and the solubility of the azide-reducing agent used. One skilled in the art will readily be capable of determining a suitable system for carrying out the invention . One example of such system is the system set forth below in example 2.

In a further aspect, the present invention relates to a peptide obtained by a method according to the present invention . This peptide may originate from a larger peptide from which it was liberated by the method according to the present invention . The peptide C-terminal to the scissile bond may closely or fully resemble a native peptide or protein .

The present invention is further illustrated by means of the following examples and figures , which are in no way to be construed as to limit the scope of the appended claims .

Figure 1 shows the mechanism proposed for the reaction :

Phosphines add to the electron deficient centre of the azide 1 initially forming intermediate 2 , that may either hydrolyze through postulated intermediate 3 into the triazene 4 or fragment into aza- ylide 5. Triazenes have previously been shown react through an S _N 2 reaction with suitable nucleophiles of either intra- or intermolecular origin . It is worthy to mention that at elevated pH conversion of 1 to homoserine without peptide cleavage occurs , indicative of S _N 2 attack by OH ^" . The protonated triazene 6 enables an energetically favorable five-membered membered ring closure resulting in cyclic imido ester 7 , a pathway analogous to the cyanogen bromide induced cleavage of the methionine peptide bond. Finally, hydrolysis of the imido ester 7 produces the homoserine lactone 8 and amine 9. This mechanism is supported by the ¹⁸ O labeling experimental results described below which allow the carbonyl oxygen to be conserved in the lactone .

Alternatively, the phosphine activated azide , aza-ylide 5 can be reduced to amine or may become protonated generating intermediate 11 which via intramolecular S _N 2 displacement involving the amide oxygen atom yields the common intermediate imido-ester 7 , that will then again hydrolyze into 8 and 9.

The cleavage induced by (di-) sulfides is presumed to be initiated by- attack of the thiolate anion at either the α- or —as depicted— the v azide nitrogen (intermediate 10) , that after elimination of a cyclic disulfide gives the triazene 4 , which follows the pathways to 7 , or can be reduced to DAB .

Figure 2 demonstrates recombinant Azhal-PYP cut by TCEP, 2- mercaptoethanol or DTT . A: Coomassie Blue stained gel of equal amounts (23 μg) of Azhal-PYP either mock incubated or incubated with 2ME, DTT, or TCEP in the absence and presence of urea as indicated . B : MALDI-FTMS spectrum of TCEP cleaved Azhal-PYP . C: sequence of His- tagged PYP from E. halophila , in which X denotes azidohomoalanine . Intact molecule mass and tryptic peptide mapping confirmed >95% incorporation of azidohomoalanine at methionine coded residues . A coverage map obtained with TCEP (above the sequence ) and DTT (below the sequence) is presented . Solid lines indicate peptides resulting from cleavage that were detected with MALDI-TOF; dashed lines indicate peptides that were detected with ESI-FTMS .

Figure 3 shows a comparison of the cleavage of Azhal-PYP (all methionines replaced with Azhal ) with TCEP and methionine containing PYP with cyanogen bromide . Digests were loaded onto an LCMSMS system. A: Extracted ion chromatograms of the doubly charged signal at m/z 921.9 of Azhal-PYP x TCEP (top trace) and PYP x CNBr ( lower trace) show less than 1 sec retention time deviation . B : Deconvoluted MSMS fragmentation spectra of the doubly charged ion indicated in panel A with annotation of fragment ions and parent ion (par) . Identical fragment spectra prove the identicity of both peptides . C : sequence of the peptide and annotation of retrieved fragment ions . U denotes homoserine lactone .

Example 1. Protein synthesis and expression

To test the general occurrence of the cleavage reaction according to the present invention in Azhal-labelled proteins , HIS- tagged recombinant Photoactive Yellow Protein ( PYP) from Ectothiorhodospira halophila was produced in methionine auxotrophic E. coli grown on L-Azhal containing media as follows .

L-Azhal was synthesized from L-BOC-2 , 4-diaminobutyric acid (Chem-Impex, Wood Dale, USA) by diazo transfer using Triflic-azide (TfN ₃ ) (Lundquist, J. T . & Pelletier, J. C . (2001 ) Improved solid- phase peptide synthesis method utilizing alpha-azide-protected amino acids . Org. Lett . 3, 781-783 ) , followed by BOC deprotection with dioxane/HCl in dichloromethane . After addition of an Fmoc protecting

group, peptide P ( sequence : PPHHHHHHPPRGFGXGFR in which X denotes Azhal ) was synthesized using standard Fmoc chemistry ( ServiceXS , Leiden, the Netherlands ) .

To produce Azhal-labelled HIS-tagged recombinant Photoactive Yellow Protein ( PYP) from Ectothiorhodospira halophila ( for sequence see figure 2b) , E coli strain CAG18491 was transformed with pREP4 and pHISP (Kort, R . et al . ( 1996 ) The xanthopsins : a new family of eubacterial blue-light photoreceptors . EMBO J. 15, 3209-3218 ) , and grown in M9 medium containing 400 mg/1 L-Azhal , 50 μg/ml kanamycin . Protein expression was induced by 1 mM IPTG for 4 hours at 37 ° C .

After lysis , the chromophore p-coumaric acid was inserted (Hendriks , J . et al . (2002 ) Transient exposure of hydrophobic surface in the photoactive yellow protein monitored with Nile Red . Biophys . J. , 82 , 1632-1643 ) and the protein was purified on Ni-NTA agarose (Qiagen, Venlo, the Netherlands) .

With electrospray ionization mass spectrometry only the protein in which all 6 methionine residues have been replaced by Azhal could be detected (data not shown) , indicating >95% incorporation of Azhal into the recombinant protein .

Example 2. Peptide cleavage with TCEP

Optimal cleavage of peptides and protein was achieved with a peptide concentration of about 0.2 mg/ml in 50 mM Na-acetate buffer pH 4.4 and 1-100 mM of Tris-carboxy-ethyl-phosphine (TCEP) . For ¹⁸ O incorporation, an aliquot of H ₂ ¹⁸ O (>95% atom ¹⁸ O, Campro Scientific, Veenendaal , The Netherlands ) equal to the rest of the reaction volume was added . Reactions were left at room temperature overnight . For hydrolysis of homoserine lactone , an excess of unbuffered IM tris (hydroxymethyl ) amine was added and left for 48 h before sample cleanup with ZipTip Ci ₈ (Millipore, Bedford, USA) .

TCEP-induced reduction and cleavage of purified Azhal-labelled PYP was analysed by SDS-PAGE ( figure 2a) and mass spectrometry ( figure 2b) . Using MALDI-TOF MS or ESI-FTMS ( for particulars thereof, see below at example 4 ) we were able to detect fragments N- or C- terminal to all Azhal residues (figure 2b) of TCEP treated protein . All fragments carried a free amine at the N-terminus and all , except the C-terminal peptide that ended in valine, having a homoserine

lactone residue at the C-terminus . This was confirmed by tandem mass spectrometry using collision induced dissociation on ESI-FTMS . Peptides which contained ^missed cleavages' all had masses which were in accordance with Staudinger reduction of the internal Azhal residues into 2 , 4-diaminobutyrate . The fact that TCEP mediated cleavage occurred C-terminal to all Azhal positions in Azhal-PYP indicated that the cleavage reaction apparently posed no special prerequisites to the residue C-terminal to the scissile bond . This underscored the high specificity and general applicability of this mild chemical cleavage reaction of peptide bonds C-terminal to all Azhal residues .

Example 3. Peptide cleavage at different pH values

The cleavage process disclosed in example 2 was performed in a broad range of pH values (tested from 3 to 10 ) . We used SDS-PAGE and densitometric determination of Coomassie stained bands corresponding to intact PYP to study the pH dependency of the cleavage and reduction reaction (results not shown) . It appears that both cleavage and the reduction proceed in a broad range of pH values (tested from 3 - 10 ) . The rate of reaction is slow at pH below 4 , due to decreased solubility of TCEP . The highest ratio of cleavage : reduction occurs at pH 4.5 - 5. Under these conditions approximately only 5% of PYP is still intact and fully or partially reduced . From this result we calculate that on average in roughly 40% of the cases cleavage has occurred adj acent to an Azhal residue and that in the remaining 60% Azhal-residues have been converted into 2 , 4 diaminobutyrate residues .A somewhat optimal plateau was observed at pH 4.5-5 resulting in equal amounts of reduction and cleavage . At higher pH levels hydroxide attack at the iminophophorane was favoured to give classical Staudinger reduction . The reaction ran to completion in 100 minutes at 50 ⁰ C and pH 4.5, judging from the disappearance of starting material .

Example 4. Cleavage of peptide FFRXGFRF

Peptide FFRXGFRF, wherein X denotes a Azhal residue, was synthesized with solid phase Fmoc chemistry and cleaved using TCEP at pH 5. The products of this cleavage were FFRO, wherein 0 denotes a homoserine lactone residue , and GFRF as a free amine .

Example 5. Mass spectrometry

Reflectron MALDI-TOF mass spectra were recorded on a Micromass TofSpec 2EC (Micromass, Whyttenshawe, UK) . ESI-FTMS spectra were acquired on an APEX-Q FTMS (Bruker Daltonics, Billerica, USA) , for low energy CID the ions were activated in the external collision cell or produced by SORI-CID in the FTMS cell.