SITE-SELECTIVE ACYL TRANSFER

Abstract: A method for site-selective acyl transfer in or amidation of folded polypeptides and proteins based on the use of a chemical structure element comprising an imidazole function, such as histidine, in position <i>i</i> flanked by at least one functional group to be amidated, such as lysine, in position <i>i+3+4k</i>, where k is an integer equal to or higher than -1, or in position <i>i-4-4n</i>, wherein n is an integer equal to or higher than 0, and at least one activating group, such as serine, in position <i>i+4+4n</i> or <i>i-3-4n</i>, respectively.

Description: SITE-SELECTIVE ACYL TRANSFER Field of the invention The present invention relates to site-selective acyl transfer. Background of the invention So-called acyl transfer reactions involve the transfer of an acyl group (the residue of an organic acid after removal of the carboxyl hydroxy group) either internally within a chemical species or from one chemical species to another. Examples are amide formation, trans- esterification and hydrolysis. It is well known that acyl transfer reactions can be catalyzed by imidazole in aqueous solution, the imidazole, which is a strong nucleophile, forming an interme- diary reactive complex with the acyl group. Also polymer- supported imidazoles have been used as acyl transfer catalysts (see e.g. Skjujins, A., et al . , Latv. PSR Zi- nat. Akad. Vestis, Kim. Ser. 1988 (6), 720-5). It has further been shown that small peptides con- taining a histidine (His) residue (an amino acid which contains an imidazolyl group) can have hydrolytic activity. Recent progress in designing structural proteins and peptides has resulted in the preparation of several pep- tides with substantial catalytic activity (W. F. DeGrado, Nature, 365, 488 (1993). For example, K. Johnsson et al . , Nature, 365, 530 (1993) disclose a short self-associating Leu-Lys-rich helical peptide that accelerates the rate of decarboxylation by means of a Schiff's base intermediate between a substrate of oxaloacetate and an amine with an electrostatically depressed acid constant (K a ) . It is mentioned that the secondary structure is important for the activity. Nature's enzymes form powerful catalysts by combining residues that are by themselves only modestly reactive. It is therefore not surprising to find that sites other than those responsible for catalysis also show chemical reactivity and that unexpected reactions can occur on the protein surface. The combination of His with Lys, for example, in a helix forms a simple two-residue site where the Lys activates the His residue in its reactions with active esters and the His activates the Lys 1,2 . The overall amidation rate of the lysine is at least three orders of magnitude larger than that in the direct reaction between the lysine and the esters 1 . Summary of the invention It has now been found that the above mentioned amidation reaction is considerably more complex and has much wider implications than first expected since the acyl groups of esters that react with His residues on the surface of proteins can migrate as covalent intermediates from the initial reaction site to form amides at the side chains of lysine residues more than 10 A away. They undergo a series of transacylation reactions on the protein surface and, as a result, migrate from histidines to ser- ines, from serines to serines, and from serines to lysi- nes in pathways that appear to be strictly regulated. In fact, the situation can be compared to a complex traffic regulation system as the pathways allowed for acylgroup transfer appears to follow rules of high specificity and directionality. The implications of these acylation reac- tions in native proteins are that the acyl groups of esters and amides, and perhaps also the corresponding ones of phosphoesters, can be generated at sites different from the ones where the product is eventually formed. The implications for the chemistry of proteins is that post- synthetic modifications on a high level of complexity are possible using folded proteins that do not need to be protected. The invention thus relates to a method for site- selective acyl transfer in or amidation of folded polypeptides and proteins based on the use of a chemical structure element comprising an imidazole function in po- sition i flanked by at least one functional group to be amidated in position i+3+4k, where k is an integer equal to or higher than -1 or in position i -4 -4n, wherein n is an integer equal to or higher than 0, characterized in that it also comprises at least one activating group in position i+4+4n or i -3-4n, respectively, wherein n is as above . The reaction between a His residue and an ester is a two-step reaction where the first and rate-limiting step is the formation of an acyl intermediate under the re- lease of the leaving group. In the second step the acyl intermediate reacts with the most potent nucleophile available to it. In the reaction between p-nitrophenyl esters and imidazole in aqueous solution, a model reaction for the His catalyzed one, the reaction product is the carboxylic acid and the reaction is hydrolysis. In the presence of 10 vol% trifluoroethanol the reaction product is the corresponding trifluoroethyl ester 4 . In folded proteins the intramolecularity of flanking lysine residues make them the most potent nucleophiles even at pH 5, where they are predominantly protonated and the reaction is an amide at the side chain of the lysine residue 1 ' 2 . The rules for the His-Lys pair were reported previously 5 and a His in position i is able to acylate a Lys in position i+4 or i -3 but not in position i -3, i-4 or i- 1. In order to investigate whether lysines could be acy- lated that were more distant, proteins were designed in which amino acid residues were introduced in between His and Lys residues to mediate acyl transfers . According to the present invention it is possible to site-selectively form amide bonds between lysine side chains and acyl groups of active esters. Anything that can be transformed into an active ester will thus be ω ω KJ N3 t→ Ul o U1 ■ O π o U1 et et r 0 H- Φ *ϋ r rt * fu μ- • Ό Ti O 0 Φ PJ CD rt Ω H Ω et μ- Ω SD Cfl Ω O Hi ii rt TS rt H- X X Φ H- μ- Ω CQ O ii φ X p rt μ- SD Φ O X ø 0 Φ ii o SD ii Φ μ- X ϋ 3 & £» Ω < υ 0 0 Ω Φ μ- O Z Ω Pi li < £ S" 3 Φ CQ H •P Φ 0 i < Φ O 0i Φ Φ rt- Φ rt Φ et P 3 0 φ → CQ CQ rt Φ ø Φ Ω • ϋ 3 rt rt O SD OJ rt ts c- CQ X CQ H- CO H P sQ tr Φ φ ω ti Ω tr rt * μ- 0 X tr Φ tr rt μ- Φ CQ H- rt CT Φ CQ P- 0 & Ω 0 φ μ- CQ Q o rt Φ 1-3 μ- μ- ø J ) J Ω 0 SD Φ T3 l→ μ- ø CQ tr Φ Φ 0 Hi μ- 0 J" 0 i 0 rt 0 ø 0! X 0 CQ < SD K rt rt φ <! < ø Φ Φ H O 0 a sQ rt Φ Φ 3 f rt *ϋ Φ 0 tr Φ SD M 0 3 3 μ- Φ Φ ii 0 Φ CQ CQ Φ Φ 0 h H CO Φ Φ Φ φ ø SD φ Ω Ω Pi CQ CQ 0 O SD tr 0 rt W CO S> £ & P Pi ra Φ rt rt ii ϋ tr et rt •P ti SD P. O • -<; Φ 0 - et tr Φ rr *ϋ X rt Φ Ω tr rr rt Φ μ- Ω Ω φ Φ Su 0 X μ- ii CQ t→ 3 X μ- t-x H PJ rt Φ H- ffi ω ft w O μ- CQ <τ} P Φ P> H 3 Ω rt 0 2 PJ μ- O 0 φ CQ Η !D Z CQ Pi O T3 φ CQ H- H- P P. (ϊ H- et P Φ CQ T5 P ^ Ω O H Pi rt ø ø rt 0 ti I- 1 rt rt ii Hi Φ 0 3 Φ Φ X <J μ- rT et tr ø et Φ 0 3 Φ P 0 φ Φ et SD ø O rt tr Φ ø et u Φ rr X 0 Φ Pi <! O tr P H K Oi Φ Hi μ- & et μ- P: tr 3 X Pi X 0 IQ rt 0 φ ϋ 3 X μ- φ P Φ Ω φ ti φ P. su Pi O CQ 0 ø Φ Ω CQ |* d μ- ct • Φ CQ rr Ω Φ Φ et P> o et μ- CQ H Ω ' CQ CQ SD φ ^ Φ P. SD Z X et CQ £ μ- X 0 • ^ O PJ rt * 0 ø 0 μ- 3 ø 0 ID O φ Φ Φ μ- 0 CQ Φ φ H H- μ- CQ Φ Φ et CT ts Φ 01 ϋ Pi X i iQ ι- χ P μ- CQ 0 ti ii X < rt 0 CQ • μ- rt CQ φ P' Φ P £ 3 Q, Φ CQ <! φ et CQ Pi Φ Φ ø ø Φ et CQ ■ P Φ φ Φ rt ø μ- Ω • P P Φ t→ et tr Ii φ W X μ- PJ φ • et rt J1J tp H P •n 0 ϋ CQ ii co X O Φ a. ii rt rt * CQ i P H rt Φ 3 Pi * - ■ O μ- P O i φ o SD SD P φ Φ 3 Ω f 0 φ X 0 φ rt 0 i-r • τs tr Φ > 0 i ! P μ- Ω < tr CQ T3 μ- *) 0 3 Ω Φ SD tP μ- rt Φ ø μ- PJ rt 0 PJ Φ O SD rt Pi rt O rt rt φ μ- ϋ Hi rt- 0 et • τs £ 0 Φ CQ Φ P <! rt !3- |P SD φ Ω μ- μ- Φ ϋ Φ 0 ii rt μ- μ- H- I- * Φ CQ ii φ μ- P μ- H Φ Pi !^ μ- Φ Φ O n; Ω tr Φ P. Q μ- ϋ ii SU O O CQ Ω Φ 3 CQ Φ Pi φ 0 H • P φ CQ μ- φ P H Φ rt μ- Φ Φ Φ t→ ii ϋ μ- ϋ P φ Ω 1-* 0 φ fu 0 et ø IQ μ- 0 μ- ø Z tr φ μ- •P rt CQ O P Φ 3 -x £ rr φ 0 CQ CQ H 0 Ω Φ sQ Hi rt P PJ ø O SD • -<: Z ii ø Φ μ- tr i t→ et ιp O ^ P φ * - ■ 0 Ω 2 Φ X i-S rt W O IP X < CQ ø * : μ- Φ K rt φ Ω *< SD Φ CQ et Ω Ω J et IQ rr φ μ- Φ 1 CQ I ** . Φ Φ T3 rt ID rt ø SD Φ O Ω CQ ii μ- 0 ) μ- 0 rt 0 ) Φ X TJ < X z μ- 3 O P ti CQ P> X 0 !^ et 0 >P o 0 μ- Φ ø rt P P < P. H- fu CQ Φ μ- T3 rt 0 ) £ i rt 0 O Ω Φ CQ Ω Ω et φ rt ii ø CQ CQ μ- "< Ω < a 2 rr μ- ^ rt Φ CQ Φ rt Φ μ- et I- 1 Ω μ- 0 SD tr 0 CQ X Pi φ Φ - rt 0 CQ Φ Φ 0 0 et n- rr rt ^ 0 Φ μ- fu O Φ 0 O y < μ- ~ Φ μ- X ii 0 0 I- 1 3 TJ ϋ rt 0 Φ μ- CQ Ω O P ) Ω ø μ- et ti μ- IP ID CQ ø y * μ- ϋ ø H SD i-d ø 0 et • "< • . Ό et Φ tT T-i & 0 ) μ- Ω 0 ) ø i rt - ø μ- P. Hi I- 1 rt Pi μ- tQ Ω - iQ Φ 3 ≥! Ω O φ Φ Z SD Φ rr CQ Φ P <! X ø Φ μ- 0 μ- Hi rt * Φ ii μ- 0 PJ ID CQ V rt ø J Hi rr 3 rt μ- Φ & Φ φ O 0 | p O 3 3 φ tr P ø ø SD ø 0 μ- rt μ- CQ C CQ rt £ H- H- 3 ϋ 3 ≤ 3 et Hi • ≤ 0 ii ii IP ii Φ rt Ω iQ rt S3-. ø O & φ ϋ rt μ- 13 rr N φ Φ μ- Φ μ- CQ 0 μ- X A> Φ tr φ rt 0 O tr ø to Φ X Pi 0 Ω H- Φ us ISI Ω rt μ- H CQ ø Φ CQ μ- Hi • n rt ø CQ tr ø CQ * Φ SD Φ Ω Φ φ 0 et O 0, P- n* Ω Φ rt * X rt rt CQ • ^ CQ Φ Φ 0 et SD 0 μ- μ- X CQ O SD SD CQ H- Hi φ fi- , μ- Φ !J h ω Φ W 0 ) 3 ø Φ et 0 ) SD Pi Ω Φ H Ω CQ Ω O Ω Ω 3 μ- <! J 0 ) ^ tr • Ω SD rt - rt t→ φ α φ l- 1 0 Φ 0 o tr Ω 0 tr J D • ξ 0 H H- O Ω Φ CQ N Φ 0 tr μ- X μ- 0i CQ CQ ø μ- CQ 0 O ii Oi ø 0 ) H- ø Φ rt 0 0 CQ Φ PJ » ) fu 3 Pi μ- Φ ιp Ω Ω Ω !D μ- et ø 0 tr ϋ H- t→ t→ Ω P ) tr P μ- rt I— 1 H (D A> Z 1* 3 ^ Φ ø 0 ) X SU Ω • P rt ω ϋ < 0J Ω • x, Pi SD P Φ 03 Ω P H φ P Ω Ω sQ μ- ø φ ø 0 1-3 O X 0 O Φ t & μ- Ω D3 ϋ ISl P Φ SD H Φ Ω Ω 0 i Ω O ; Φ !D Z 0 Pi 3 M t ii Φ φ t→ ø • X3 ϋ P Φ Φ Φ Φ et P 0 O rt £ O 3 rt rt μ- et < CQ μ- tr Φ 0 ( l→ et rt * SD IQ tr ϋi P, 3 H et et μ- CQ μ- 3 <X3 Φ rt X Φ • Ω Φ μ- SD Ω Φ μ- 0 ri rt O Φ Ω CQ SD tr SD PJ *1 Ti C <! rt 1 t→ 0 ) •» ■ P X Φ 3 su 3 tr Ω O rt & O rt 0J φ rt 1 et 0 rt c ϋ OJ 0 ^ μ- Ω Φ ø K rt t→ SD ω μ- tr 0 tr 13 Φ 0 O H- Φ ii H- et IQ 1 ϋ Φ rr O X P. fl) J 0 ) μ- Ω ø 1 0i :> μ- CQ P. 0i 0 O Φ 0 Φ J ^ 0 P σi Ω O 1 rt CL, rt rt S ø 1 φ 0 1 0 1 1 J 1 ø O 1 0 ^ 1 cause the different geometrical relationship between His and Lys residues gives rise to acyl transfer reactions that are not equal. The experimental conditions described for the present invention have been standardized for com- parison between different sites and can be further optimized. The introduced substituents are also known to influence the incorporation at a neighboring site because of its size and other molecular properties, such as for example charge and polarity. When the protein that has been bound to a surface by a covalent bond is a homodimer then only one of the peptides is covalently linked, and the other peptide can be replaced to form a heterodimer. The functionalized protein is readily removed from the surface and replaced with a second one in a very * short period of time (a few seconds or minutes) , so that a new reaction can be performed. Examples of targets are proteins and other bio- molecules that are extracted from, e.g., cell lysates. Combinatorially composed surfaces can then be used in the search of the "unknown proteins" that are not readily predictable form the genes in the cell, perhaps because they have been posttranslationally modified, so that their function is not predictable from the DNA sequence. An added value is the capability of constructing tailor-made affinity columns form purification and isolation. The receptor or binding site that were used for example to find a specific protein from a cell lysate can be constructed in large enough quantities to be used for its purification and isolation. One object of the present invention is to provide an improved method of performing an acyl transfer type reaction using an imidazole based catalyst. There is therefore provided an improved method of performing a chemical reaction involving an acyl transfer mechanism in the presence of an imidazole-based catalyst which can form a transition complex with the acyl group. ω ) DO t I- 1 μ> ( Jl o ( Jl o ( l o uι Λ Ω z <! Ω ■ P μ- Ω α TS 03 rt 3 X ■ P 3 rt CQ μ- Ω 03 •n SD 03 w rt • X3 03 Ω ø 0 X Φ O 0 H SD 03 0 s ϋ rt ø φ 0J Φ Φ tr •P Hi 0 rt H Hi rt I- 1 tr O t SD Φ 0 Ω ti 0 rt 3 α μ- ϋ et rt <! <τi rt Φ φ l→ i O ii φ ii 03 ii t-x 0 et Ω rt Φ μ- <! rt μ- 03 ø Φ tr μ- et X Ω CQ φ ø < 0J 0 X 0 μ- 0 Ω SD X 0 ø μ- Φ rt Hi Ω φ Ω CQ O Hi μ- O SD μ- 0 ^ Ω μ- Ω Ω φ 0 rt Ω 03 Φ H μ- 4 >υ iQ ω H 0 IP 0 03 rt iQ iOi • tr Ω H rt P. ^ rt H 03 sQ μ- rt rt i→ 3 y Ω f Φ rt Hi ø O Φ Hi 0 μ- tr 03 Hi ø Φ t- 1 0 Hi • X O ø ii ø 01 μ- o SD 0 rt μ- Φ 03 03 rt H H 0 ID O O < Ω 0 ii Pi i 0 ii 0 t-x SD Hi Hi 0 <! X SD ii X SD rt ii Φ Hi Ω rt Φ !D SD Ω O Φ et Φ rt et Φ Ω ID CQ ιp 03 μ- Φ 0 • P μ- ii SD Ω 0 t tr Hi OJ X X et rt 0 ■ ri rt & <! 3 ϋ P. ø 0 Φ O • x) μ- ø Ti φ μ- Hi Φ z 0J Φ Φ Φ X tr 0 T3 Q μ- SD φ 0J φ ID φ Ω 03 t→ 3 M 3 03 ϋ H ø ■ ; Φ SD μ- Ω Hi t→ 0J SD i ii Φ Φ rt rt 03 tr Ω Hi rt 03 ■ P rt Φ • P 0 μ- rt 0 et μ- 03 Φ rt- tr 03 Φ 3 SD rt <! Φ Φ 3 ii ø α 0 rt 0 X CQ ϋ O 3 H μ- fli 0 Φ iQ g tr Φ 3 Φ Ω μ- Ω φ ø ct s 01 <! 0 rt Φ 0 Ω ϋ Φ μ- 0 SD rt £ tr ϋ i=: tr 0J ** 3 φ Φ rt • μ- Ω Φ Q ø Φ Φ 0 ii φ ii X CQ et O 3 Hi Ω N ø Ω 3 Hi tr o 0J μ- ii Hi tr l→ rt ιp ι- χ ii rt 03 fi Φ Ω SD φ 3 μ- rt φ μ- 0 et μ- φ Φ Ω 0 •n & Ω rt 0 Φ ti Φ φ C CQ φ i Ω ii ø μ- μ- O ø CQ Hi t→ μ- ø Ω & rt H Φ μ- • P OJ t et Ω 0> 3 Φ CQ ii o Φ Ω 0 Hi ii ιp OJ tQ φ Hi ω et μ- μ- μ- v 3 φ Φ z ii 0J Φ Φ ϋ tQ 0 O 3 O et μ- 03 SD CQ SD O Hi et φ P SD o SD HI Hi SD φ 0 j3 rt • n tr ø •P O 03 SD ø 0 ) l-h tr O ϋ ø tQ I- 1 - Hi ϋ CQ Ω et Hi 0 rt φ 3 Φ Ω Φ o Φ n CJ. ti ϋ ϋ μ- 0 O ^ ^ 0J iOi rt 0J rt rt X CQ tr 0 ■ g CQ tr ι- χ O Hi μ- μ- μ- φ Φ O ø πr Φ • P 0 ϋ I- * • Φ ^> ø ϋ μ- rt Φ Ω Ω Q *U 0 Φ 3 Ω Hi Hi 03 Ω rt •X3 iQ 0 3 H Ω CQ >ϋ Ω μ- 0 et 0 O tr Φ rt O 0 Φ 3 V Φ SD μ- et Φ Φ 03 μ- μ- O rt Ω Φ O N 03 O rt Ω ø 3 Φ ii 03 μ- Hi SQ-. Ω μ- φ H Hi O Hi μ- •P ) rt Hi rt μ- O P Φ 3 Φ μ- ti X μ- rt •S μ- O 03 μ- μ- 3 ø μ- Φ rt ii IQ 0 ø rt Φ SD Φ O 3 φ 03 TS rt Φ 3 ø μ- h Ti <! rt Hi rt 3 Φ et CQ Hi μ- <! 0 P. tsi μ- Hi TS rt φ μ- < τ3 μ- ti 03 O ii μ- Φ OJ μ- Φ μ- Hi Φ Ω α *ϋ ) 0 Φ ii 0 Φ rt ø H μ- 3 φ rt O ϋ CQ Φ <! φ 0 ) 1 rt Hi Λ Ω rt SD 03 0 ≥i Φ Ω iQ SD l-> μ- Ω rt X tr Hi O rt 0J ø Φ CQ CQ 0 rt u ' CQ (Q-. W Hi X et !D tr φ 0 H CQ φ n rt H N Φ 03 Φ rt >P IP rt 0 SD μ- rt ø Φ Ό 0 Hi 0 ti rt μ- φ μ- 03 0 rt Φ ø 0 I-" Φ HI X φ H Φ O 0 Hi Hi Ω μ- Ω φ Ω H μ- <! Hi Hi 0 • s=: X μ- Ω l→ Φ Ω rt Φ Ω O CQ 0 φ ιp 0 Pi M i p •n 0 03 Φ 0 V rt IQ IQ < sQ 0J ø rt Φ 3 i μ- rt 0 Ω μ- Hi φ Φ H rt t→ SD 3 •» 0 Φ X μ- φ X rt £ Φ T3 μ- 0i • P O rt rt X μ- 3 SD CQ μ- Φ μ- et ø a T-5 Φ 0J Hi ø ii Hi ι-3 SD ø tr μ- ii X μ- Φ 0 μ- rt Ω 03 sα ø Z z et rt i Φ rt et μ- Ω Φ 0 rt tr tr < 0 Ω μ- Φ 0 Φ TJ H ι-3 Φ φ ii μ- X μ- μ- Φ 03 φ μ- tr rt O 0 φ μ- φ <! SD Hi μ- ø <! SD ii ø X 0J Hi • » μ- SD rt μ- O rt M 0 Φ 3 Φ Ω Ω i Hi Φ t→ Q 3 PJ Φ N 0 Ω Φ Ω et ø tr Ω Hi Φ 0 ii μ- P Ω | p rt O φ μ- rt t-x 03 Φ Ω SD 0 et rt μ- 0i tQ α X 1 ii ø Hi 0J 0J ϋ Φ μ- rt 3 rt μ- μ- "< 0 • τi rt tr I- 1 Φ μ- μ- Hi SJ OJ Ω Z Φ Ω IQ μ- Ω ø μ- 3 O Η μ- • * <; O Hi Ω SD X IQ 0 0 φ μ- Ω 3 0 Pi rt > 3 O X rt CQ •• < 03 Φ Hi ii 03 Hi rt Hi X ii O H H < πs O < 03 < X ti φ Hi I- 1 - < τ5 φ Φ μ- ø PJ I- 1 tr μ- Hi μ- O Φ c rt O ** φ j≡! φ Φ 03 Φ rt rt Ω ϋ ϋ rt Φ ø rt 03 rt 0J ii 0 SD £ • ø o Tj Hi lP Hi 03 Φ Ω X SD X O Φ "P O et CQ μ- X 3 Ω O Ti rt 0 rt ø rt 0 X • P o O μ- < h H Q μ- Hi Φ Ω Ω Φ rt Hi rt X Ω μ- μ- Φ μ- 0 £ ***** P, P Ω μ- Φ O T3 ^ μ- SD 03 IP h SD X μ- Φ tr Φ rt < X3 03 O ø 0 ID 03 φ " * μ- O α rt ii 0 rt Φ rt φ μ- 0 μ- SD Φ 0 μ- Φ μ- α Hi Ω Hi rt rt Hi μ- Ω Hi Hi Φ Φ Φ O 03 Φ SD tr SD 3 SD Hi IQ 0 ■ p Hi Φ μ- Ω Hi O p 03 CQ 3 μ- < I 0 i O l→ μ- H ) tr t→ Ω SD § Hi rt IQ rt μ- ø O Φ SD 3 rt Φ Hi t-x- Φ ø 3 Ω ii Ω tr SD tr > 1 μ- X Φ w tp Hi 1 μ- 0i ø P-. ϋ Ω O Φ N SD μ- o ii Φ 03 OJ Φ HI ιp Φ rt I et O Φ rt tr μ- Ω N li iQ 3 0 03 Φ ii 1 et rt OJ Hi H μ- Φ 0 πs 0 φ μ- φ μ- Ω Φ Φ 0 tr Hi 1 ID μ- O 1 HI iQ 3 Hi ii 1 tr 1 1 Φ 1 0J Hi φ 1 ιp a neighboring position to the imidazole function that the functional group can be site-specifically functionalized through acyl transfer catalyzed by the imidazole function. Thus, the present invention is based on the concept of increasing the imidazole type catalytic activity in acyl transfer reactions by providing the imidazole function on a backbone structure with a pendant flanking group (or groups) or chain on one or both sides of the imidazole function, which flanking group or groups can interact with the imidazole-acyl complex formed such that the transition complex is stabilized. The reaction rate for the desired acyl transfer reaction, such as an amidation, trans-esterification, hydrolysis or thiolysis, will be increased considerably thereby. While esters are the currently preferred substrates, e.g. amide and anhydride substrates can also be used. The term "imidazole function" is to be interpreted broadly, and is meant to encompass any imidazole-based structure that possesses the desired catalytic activity. The imidazole group can consequently be modified in various ways. An advantageous imidazole function for many purposes is based on the amino acid histidine (α-amino-4- (or 5) -imidazolepropionic acid) . One or both of the available carbon atoms of the imidazole function can, for example, be independently substituted with alkyl or halogen. The imidazole group can also be substituted in 1- position with alkyl. Alkyl has preferably 1 to 6 carbon atoms, especially 1 to 4 carbon atoms, e.g. methyl or ethyl. Halogen includes fluorine, chlorine, bromine and iodine . The flanking group or groups can comprise a link or chain of, e.g., 1 to 6, preferably 1 to 4 atoms, usually carbon atoms, connected to a terminal functional group or other group capable of the required molecular interaction with the acyl transition complex. In case the catalytic structure element is a peptide and the imidazolyl function is part of a histidine residue, the flanking chain or chains can be pendant proton donating parts of other amino acids, e.g. selected from lysines, ornithines, arginines and/or further histidines. The chemical structure element supporting the catalytic imidazolyl function should preferably have some type of rigidity, such as secondary structure, in order to localize the flanking group or groups with respect to the imidazolyl function in an optimal geometric relationship for the desired transition complex-stabilizing interactions to take place. In an advantageous embodiment, the chemical structure element is a so-called designed polypeptide with a stabilized secondary structure, e.g. α-helical coiled coils. Designed helical peptides are, for instance, described in J. W. Bryson et al . , Science, 270, 935 (1995) . The structure element is, however, not limited to a peptide. On the contrary, it can have a variety of compositions readily apparent to the skilled person in the light of the present invention, and can thus be included in or be part of other types of structures, such as a carbohydrate, a natural or synthetic polymer, etc. The size of the chemical structure is not either limiting, and it can, e.g., be a peptide of as few as, say, five amino acids. As to the required geometric relationship between imidazole function and flanking group or groups, a functional arrangement can readily be designed for each particular situation by the skilled person after having read the present description. Depending on the functional moiety of the complex- stabilizing flanking chain or chains, the transition complex can react with such a flanking chain in an intramolecular reaction. Such an intramolecular reaction can be used for selectively functionalizing peptides, pro- teins and other molecules. It is readily understood that designed polypeptides embodying the present invention, such as those mentioned OJ tr rt SD rt rt rt • P rt 0J rt ø t→ rt rt >υ rt φ X et N et Ω O Ul rt CQ Ω Ω rt Hi SD rt ø μ- rt μ- ø O Φ tr tr l Φ Φ Φ fi X CQ ^ μ- 0J μ- s Hi et 0 Φ μ- I- 1 SD tr Φ tr Hi < Φ SOi fi 3 OJ Ul φ φ Ω O μ- o Φ rr <! rt O Hi Φ SD ii O 0 HI Φ rt 0 Oi Hi φ α φ Φ 03 rt rt O et l→ ø Ω Φ 3 ro μ- Hi tr Pi Hi 03 μ- < Φ ^ 03 03 rt μ- 03 μ- * * 03 φ fi ii o t-x O tr rt μ- φ Ω φ 03 Ω φ Ω H3 CQ Φ p, Z m tr ^ < rt 03 Φ P- ■ <; ø ≥ Φ O 0J 1-3 Ti 03 X ~ 0 X μ- μ- Ω OJ ii φ X Φ 03 O Φ μ- ) CO Ω Hi μ- 0 μ- Hi tr ii O μ- Φ tJ 03 Φ rt rt J ti μ- Φ rt fi Ω HI φ O 0 Hi μ- μ- 0J tr N Ti Φ O rt li t-x Hi Ω Ω 0 φ Hi Φ Ω 03 0 Φ μ- 03 CQ 0 ^ ø 03 ø Φ μ- 0 tr rt X l-x CQ SD Φ t-x •ϋ Ω < i Ul 3 0 ) 0 P • HI Ω μ- et O ø l-x Φ SD Φ φ Φ φ μ- Hi 0 0 Φ 0 SD tr SD tr ø 0 Φ φ OJ Hi et Φ >P μ- rt ø O rt *ϋ et Hi CQ • -<. tr μ- X Pi Hi et et •P HI Φ b-x μ- et O J rt rt IP Φ ι-3 rt μ- CQ X 0 X 0 Ti ø 0 ø Ti SD Φ tr ii μ- et 03 ø X et Ω μ- μ- O ii <τ5 tr μ- O O Φ ϋ φ 0 φ l <! Ul fi 3 Ti φ Φ SD 0 μ- SD ID μ- 0 Pi Φ rt rt Ω tr SD rt Φ O Hi rt fi et ϋ Ω •υ Φ Φ φ • P Φ fi rt Hi Pi Hi ø f 03 03 μ- O O c μ- ^ HI tr O μ- OJ rt SD et Pi Z Ti hi μ- Ti μ- Φ 03 et Ti 0J μ- ø O CQ Ω 0J Φ rt Hl 0 μ- 0 P. X OJ φ 03 Hi ii 0 OJ Φ 0 fi 0 Hi 03 fi Hi 3 rt •** et rt Φ Ω μ- O Φ et et 0 O rt Φ μ- ϋ ^ 0 CQ ^ o Hi rt rt Hi 0 Ul Ω <! H ø μ- 03 SD ø SD Ω μ- hi 0 HI Φ O 03 Ω OJ Hi P. 0 CQ tr i 03 φ O μ- Ω O Pi • P O O . ti ii 0J Hi O 0 ) Ω OJ 0 03 Ω tr rt tr . 0 rt TJ μ- φ ø μ- « fi <! SD et > ϋ 0 HI μ- tr hi 03 Pi 03 rt l→ CP μ- >i μ- Φ OJ Ω tr M tr ø Ω tr φ 0 Φ O H φ 3 o tr ø • P μ- μ- Hl μ- Ω 0 Hi en φ OJ Φ Φ Φ t-x ≥i rt Ω O fli O 0J X Hi 3 tr 0 SD Ω Φ O N CQ H rt tr 0 0 ti Pi ø rt σi Φ φ Pi φ o X ø OJ rt ø Hi ø Φ X ø tr Hi rt Ω ø Φ fi 0 ϋ Φ ø Φ Ω fi 03 • <! SD ø l→ Ω ^ 0J 0 α • < . μ- rt SD i O O rt tr Z tr tr tr 00 ø φ μ- μ- φ et Pi CQ rt et Ω Hi O fi O I- 1 ø OJ fi J ^ Φ • - SD (-π ø ip CD I- 1 Φ o ii X μ- SD J OJ Ω I- 1 SD ii < τi HI O rt Ti Hi ø 03 μ- fi ι- χ rt 03 Ω 0 μ- Z H ø Φ O fi Φ 0 rt OJ φ CP X ^ -. Ω Φ ø hi H t-x Φ ii J rt Ω Φ ti ID tr tr IP ø tr Ul CQ φ rt • O fi fi 03 μ- Φ I- 1 Φ Φ Ω Φ tr HI μ- 0 ) Pi Ω 3 ii t-x rt φ 03 ϋ 0 Φ O rt μ- 03 φ et Hi 03 O O - — Hi φ ø Ω Pi 0 tr Ω κo Φ Pi 0 rt φ μ- SD I- 1 X O SD tr Ω X Ω μ- μ- 0 0 rt 0 Ω Φ 0 O Hi 0 Φ ø OJ rt ø Φ 0 t-x 0 • rt O Φ ID • P μ- ϋ Ω rt ϋ ιp HI ϋ Φ rt μ- ***** rt et μ- 3 Hi rt SD t-x 0 et 0 Ω 0 03 fi Φ ii ι- χ πs Φ SD Φ tr Φ OJ Z μ- cp jj * O lP Q * tr ^ X tr O H φ O i TJ O μ- - SD Q hi 1 μ- Φ 3 <! X μ- tr ø μ- φ φ 03 ιp φ SD Φ et Φ 3 O <! SD ti rt 3 • P • tr 03 ø rt O μ- tr πs M Φ Φ P 03 N rt OJ Ω Pi SD 0J rt * o Φ 3 I- 1 - 0 O Φ X l-> 0 rt O OJ Ti CQ SD rt Φ • ii HT μ- tr Ω rt Pi Ω Φ rt Ω ø 0 SD φ h3 tr SD Φ φ 0 • > fi ø 1 P. Hi μ- μ- P. rt μ- Pi • τS φ rt 0 Φ Ω Ω CQ Φ rt ø Hi 03 X •* <! Φ Hi O Ω Ω μ- et ω φ OJ rt 3 ϋ μ- 03 0 μ- CQ OJ μ- μ- O rt μ- Ω 0J 0 rt O Hi M 03 fi OJ 03 μ- Ω rt H I- 1 hi O μ- ϋ ϋ Hi Ω Hi >τ3 CP 0J μ- μ- 03 ti rt SD Ω ι- χ μ- CQ et 1 Ω φ rt 03 α rt μ- μ- φ a φ Hi ø SD tr φ SD fi φ 3 3 O 0J μ- T3 Ω SD O Φ to fi ≥! Φ • P Pi 03 9 ) et o 0 O Hi 3 • P ø μ- rt <! Ti O hi 0 rt K Ω oo μ- Z > φ OJ μ- μ- Φ Ω Ω f SD tr P. O rt μ- OJ O 03 Φ Φ fi ø 0 ) tr μ- tr to tr Φ 03 μ- rt Hi Pi J rt O O rt • Φ et t→ Φ Ω rt fi et CQ O et fi φ ω Hi I- * et rt 03 Φ Φ s=: rt μ- Hi i μ- p. <! tr μ- μ- O rt O OJ m ii 03 Φ μ- 1 0 o Pi Φ H φ Q Φ μ- <! SO-. 0 O ii φ φ μ- ø rt ϋ OJ Ω 03 ø fi SD Φ SD rt fi ii t-x (^ Ω 0 ϋ 0 μ- μ- 03 φ μ- Ω Hi J H Hi Ul • Hi Ω Ω μ- ø μ- 0 ^ Ω • ; Φ Φ o t tr X tr 3 m 03 Φ rt Φ • rt 0 Φ CP rt O O rt Ω iD 03 rt OJ m Q Φ ø HI μ- <! 0 Φ μ- φ ■ p Hi O OJ Hi 3 Φ rt rt Hi μ- μ- Ω μ- • < Z fi O 0 rt 03 φ rt ti fi CQ Hi O ii Ul 3 0 fi Hi fi rt V 1 μ- φ Φ O O Hi rt SOi X φ Z l-x tr Pi X Φ rt O Hi Φ • Φ fi ui O Pi O ID CQ O ii rt ø μ- s=; 0 ι- χ - μ- ø 0 Φ SD Φ CQ TJ f ø 03 03 Hi Φ μ- 3 • P μ- Φ Hi Ti Φ O Φ Ω πs CQ so, μ- Φ Φ ø CQ μ- H rt rt fi O Φ Hi 03 I- * SD 0 tr tr . Hi CQ X SD ■ - . Ω i Hl !D 0 TJ Ω tr O P. rt tr Φ <τ3 O rt tr Φ I- 1 l 0 φ et CQ • Hi 0 Φ Ω Φ 1 1 0 iD Φ 1 ii 1 tr μ- 0J φ Ω μ- μ- et X Ω — 0 P. ^ — , Pi 3 Pi 0 ) 1 Pi Φ Ω Hi 03 O φ ø ø Pi 1 1 1 rt Φ Φ 1 μ- H ( P 1 0 03 OJ Pi Φ 1 ϋ X Hi CQ rt et Φ φ Hi 1 φ 1 1 O 1 fi Ω CQ Ul Z 03 X J 03 O ffi tr ø tr ø Z 03 Hi 3 φ Φ Φ et tr rt et O 03 Ω O 9> 0 rt TJ Φ OJ fi 0 SD et μ- 0J 01 i -d Φ Hi 0 ) Ul rt ii Φ 0 X X Hi μ- Φ tr tr rt O fi ø Hi tr OJ 3 0 0 rt rt SO-. ϋ ti Ir" Φ rt 03 μ- l-x ϋ O Hi Ω Ω Hi 0 Φ OJ 0J HI α a. P. Φ hi 0 ø μ- Φ I- 1 - Φ fi rt * 0J O Hi μ- ø t-x SD 3 rt Ω Φ Φ φ 0 DO rt rt HI Ω Φ 1 μ- <! tr Ti Pi fi 0 O μ- t→ tr cp rt 03 O 03 03 Ω in fi tr rt Φ ii et O Ti 03 Φ Φ • •> > Ω 03 rt rt Hi 0J 03 Φ J Φ 03 • g 03 03 rt 0 dp Φ fi φ Hi X hi Φ 0 & Ul J hi Φ *** <. μ- O SD Ul SD SD Φ rt fi φ O *1 μ- Hi • OJ Φ μ- rt li Φ P- TJ Hi 0 rt tr μ- rt Φ I- 1 CQ O Ω rt l→ Hi tr φ tr ■ P h μ- tr 0 < Ω 0J tr 03 fi SD Φ rt tr Ul fi OJ rt Φ OJ φ μ- ø O rt ø t→ Φ Pi 0J SD X ø Φ Hi Φ 0 tr: rt Ω 0J 0J rt tr hi μ- tr Φ φ OJ ui fi l Ω 03 μ- tr 0 Φ hi Ω O 03 ii Φ OJ μ- 0 0 μ- rt Ω ω rt Φ SD Φ Hi rt a Pi φ Ω Hi φ rt . ø rt ϋ 03 SD et 0 03 3 0J rt πs σi T3 Hi z O 03 X • μ- Ω ii φ rt 0 Φ OJ 93 03 μ- CP ID Ω rt X Hi SD μ- tr rt ti μ- CP o 03 0 φ Hi cp O Ω X SD Z fi Hi IP TJ ti et H] μ- O ii Φ Φ 03 3 ø Φ φ Φ O Φ 0 ! Z fi Hi O CP rt Ω Φ φ 0 μ- Φ t-x- μ- μ- tr tr Hi Hi μ- P. 0 φ SD Φ SD Hi Φ o\° 0 Ω φ ω I- 1 - o Hi hi Φ 0J Φ fi φ TJ tr < rt rt μ- Φ tr 3 Hi Hi X Ω 03 rt i • et 0 O 03 0 rt ti rt CQ rt Φ Φ • • 03 rt rt rt Φ • φ TJ rt rt Z rt μ- 0 μ- so X 0 l-x Hi rt 0 Ω OJ tr SD μ- t→ Z X Φ 03 a fi I- * Ω 0 μ- μ- μ- 03 O \ P- o 0J 0 O I- 1 rt φ Pi μ- 'X, 0 Hi Φ Φ ø rt μ- O *< O Φ <! rt 3 HI SD ^ - ^ rt 1 t→ Hi Pi Hi ^ tr t-x ^ Φ ø -> Hi SD Φ φ tl SD Ω Hi rt μ- X OJ ø μ- 0J 03 M X ii in rt CQ N Φ • n • 0 et Ω Hi O O et Φ Φ CQ CQ μ- rt rt l ii l-x + Φ Φ | 3 ii rt Φ Φ Ul ϋ 03 ϋ μ- rt rt TS ti 03 X rt Pi O 0 Ω μ- Φ Φ φ 03 μ- rt ω 0J 03 § μ- φ ID Pi TJ Ti ^ 0 Hi X tr rt φ ii X ø 3 Φ X a- Pi rt N tr • TJ Ω 1 • - CQ SD HI 03 et 0 Φ ø Pi Φ Pi ^ μ- CQ l Φ μ- fi μ- Φ φ 03 ιp 1 φ φ v > Φ rt Ω rt ϋ φ μ- φ X et ø SD O 3 0 tr 03 ϋ Φ ø ϋ X φ Hi μ- O — ' J μ- et o rt Φ 0 i 1 X Ω μ- Φ fi 0 Φ Ul μ- so, et J O fi fi ø ii ii II rt O X μ- 0J SD Φ CQ πs CQ o 3 O fi et SD O SD φ μ- O T3 Φ o μ- a. φ ii 3 Φ P, Hi SD φ o O Hi Ω 0 Φ Ti 03 P. 0 Ω Hi φ 0 ) ø 03 03 fi Φ 3 et Φ Φ SD • & u> Hi i rt μ- 03 I- 1 μ- tr o 0 ■• <. μ- p Φ 0 et 03 μ- rt ø X fi T. Ω αo Φ Hi TJ sD μ- Q μ- φ 03 μ- 03 ϋ O 03 Hi Φ Φ 03 tr Pi X Φ 3 tr Ω rt μ- rt rt H SD 03 rt O μ- H Pi - πs rt Ti rt μ- Ω 03 ^ 03 φ Φ Ω φ φ Φ X 03 i μ- μ- 1 μ- X Ω Φ s >τi X Hi ti ° Ω ø ø rt ii 0 03 O rt 0 rt rt rt 03 O Hi Φ SD Pi O 0 CQ Φ rt 0 1 o Φ 03 φ OJ Ul 0 OJ ϋ μ- ii φ ii tr O fi Ω 03 Hi rt Φ Hi Ω rt Φ ts fi 0 ι- χ ti rt φ Hi I- 1 - et SD ø 9> Ti ti Φ 0 ) fi Φ i ffi l hi et 0J Φ rt hj o Ti Ω 1 OJ Z 0 rt Φ rt ø O CQ tr rt μ- Ω "> et O X 03 Pi Ti rt tr fi l Φ Φ SD O <! X OJ IP tr μ- 0J P, hi CQ 0 TS φ Φ tr O Φ Φ 3 Φ φ Ω Φ ii tr Φ 9> 0 Hi 1 X TJ ti Φ φ Φ 0 Hi SD 0 3 Φ Φ Hi 03 0 ) ii O - O Ω O φ ø Ω Hi μ- H ID et Ω Pi Ω l-x 0 03 Hi ^ μ- rt tr Φ TJ Hi μ- Pi & rt Hi Φ ϋ 0 ii Ω rt H 3 t-x- 0 Φ tr ■ " ii 03 rt ø μ- 03 rt ø 03 φ 03 X Φ rt l Φ Φ φ 91 μ- ω 03 TJ Pi fi Hi - rt φ Φ Z Ω O ft T μ- Hi 0J 3 ii s=: TJ rt tr et 0J Φ X et O • rt rt Φ OJ ii Ω tr P ii O 0J O M O Pi Ω OJ tr O Z tr Φ μ- Φ SD 03 i Ω Hi ID 1 X φ et hi 0 OJ Ti 0 s=: 0 ^ ti ID CQ Φ rt Pi 0 03 μ- tr 03 fi Ω 0 Φ CQ Φ et O Φ » Ω Φ 0J 3 Hi fi cp φ μ- Ti rt μ- μ- Pi < rt 03 Φ rt μ- 0J 3 et 03 03 tr O ii Hi O Pi rt 0 0 Hi 03 rt i O tr φ rt μ- O Φ Φ Hi fi 3 SD • rt CQ φ Hi 9> iOi μ- Hi Φ rt tr rt Hi Ω X Φ ϋ Φ μ- 03 Hi Pi hi 0J 0J Φ cp ■ D tr ϋ Φ OJ i- Ω Hi tr Φ Φ OJ φ μ- Φ Ω SD Ω 3 O 0J Φ Pi OJ rt ø Ω P HI φ Φ tr 03 CQ fi rt Φ ϋ Ω Φ Ω ι- χ Hi ti rt SD SD HI J SI SD Φ et 0J μ- tr Pi 03 Φ 0 Φ rt Hi O O 03 rt 03 Ω Φ μ- 0 03 ø μ- 9i tr Ω Φ t→ 03 9> l-" rt Φ Ω rt cp tr fi fi SD πr rr 3 ø ii Φ μ- TJ 0 0 O SD O 03 ti N 0 Φ rt P. t-x Ω s=: fi rt 0 Φ μ- 03 OJ Φ fi l Φ t- χ 0 ii fi 0 CQ Hi φ tr φ μ- Φ i Hi μ- et 0J 0 Pi φ tr ts fi Hi rt rt rt J 0J rt rt 03 Φ HI 0 μ- 0 et Φ φ X Hl Pi z X fi 3 I- 1 Φ Hi 0 Ω HI fi tr Φ Φ 03 rt X O μ- Ω rt ø tr ii Hi rt Ω μ- 03 rt Φ X Φ Φ Φ 0J 0 Ul φ 03 μ- SD 0J Ti Φ μ- Φ HI 0 rt CQ Φ CQ t→ SD et SD X Hi Φ φ tr O X μ- 0J rt 0 ii Hi X ø rt Hi Ω et Ω O rt • μ- ii Ω Φ ii X <l Φ Φ CQ P. 0J OJ 03 Ω 03 X CQ μ- Φ et rt OJ cp Φ O μ- o t5_ Q < 0 et rt - * <! Φ O Ω 03 < O rt μ- tr ø 0 !=: O tr ii I- 1 rt Hi P- HI Φ O SD Φ ø μ- tr μ- ϋ φ Φ tr Φ Hi rt tr O ii Hi rt J φ SD t-x O tr 1 Φ Pi Ω Hi 0 l→ Ti 0 Φ ø 03 03 CQ ■* <. Φ 1 Z Φ tr 0 1 OJ 1 1 K * . Hi 1 - ø 1 Φ rt the lysine can be reacted with the hydrophobic substitu- ent using standard carbodiimide coupling reagents. Alternatively, some substituents may require HPLC purification at an intermediate stage of the functionali- zation, if the chromatographic properties of multifunc- tionalized proteins do not differ appreciably. Alternatively, some substrates may function better at different pH values of the buffer. 2 , 4-dinitrophenyl esters will, for example be incorporated very efficiently at pH 4-5 due to their inherent high reactivity. This will allow the introduction of several esters simultaneously into the reaction vessel, and the reaction is then controlled by a change of pH. For example, a mixture of three esters is added to a peptide, which is held at pH 4. One ester is a 2 , 4-dinitrophenyl ester, which reacts readily at pH 4 and is thus incorporated at the most reactive Lys. Then the pH is raised to pH 5 and the N- hydroxisuccinimide ester reacts and is incorporated into the second most reactive Lys site. Finally the pH is raised to 6 and the p-nitrophenyl ester is incorporated at the third most reactive site. Alternatively, the incorporation is performed at the surface of a solid support, e.g. a gold plate in a Bia- core instrument. Then the first reaction is used to form a bond between the active ester attached to the surface using e.g. thiol derivatives that are well known to bind to gold, and the most reactive Lys. Then an ester substrate is introduced into the reaction chamber and reacted to form an amide at the second most reactive site, and so on. Brief description of the drawing Figure 1 is a modeled structure of a monomeric hairpin helix-loop-helix motif showing the positions of the residues that form the catalytic network; the sequences are those of the peptides described below; the arrows in- dicate the acyl migration pathways from His-11 to Lys-7, Lys-10, Lys-14 and to Lys-34. Detailed description of the invention A model protein, the 84-residue helix-loop-helix ho- modimer described and characterized in detail elsewhere 6,7 , Figure 1, was redesigned to have Ser residues i- 3 , i+4 and i+8 relative to a His(i), and with Lys residues in positions that could not be acylated directly by the acylimidazole intermediate on the His side chain. The resulting 42-residue peptides, given in table II below, were synthesized on a PerSeptive Biosystems Pioneer automated peptide synthesizer using a Fmoc-PAL-PEG-PS polymer, standard commercial protocols and F oc protection group chemistry, purified using reversed phase HPLC on a semipreparative Kro asil column using 36-38% isopropanol in 0.1% trifluoroacetic acid and identified using elec- trospray mass spectrometry (ESMS) . The mean residue el- lipticities at 222 nm and 300 μM concentration were meas- ured and typical values were -19000 deg cm 2 dmol "1 . The geometric relationship between the amino acids is shown in Figure 1. The proteins at 1 mM concentration in aqueous solution and pH 5.1 was reacted with an excess, typically 40-60%, of mono-p-nitrophenyl fumarate (I) . The ex- cess amounts of substrate were needed since background hydrolysis wastes some of the ester, and the excess amount was estimated from the relative magnitudes of the second-order rate constants 8 , Table 1. The resulting proteins were analyzed by HPLC, using analytical columns, and by ESMS (MW of the monomeric peptide S-I for example is 4333, found 4333, and that of the corresponding mono- fumarylated peptide is 4431, found 4431) , with and without prior tryptic cleavage to identify the site of amidation as described earlier 2 . The fragments expected upon trypsin cleavage are well defined and informative as the peptide is cleaved on the C-terminal side of basic residues, unless the side chain has been modified by fu- marylation in which case it is not cleaved at that residue. Table I. Second-order rate constants for self-catalyzed reactions of various peptide with mono-p-nitrophenyl fu- marate at pH 5.85 and 298 K. Peptide κ s i/NM- 1 ) S-I 3 .9xl0 ~2 S-II 3, .9xl0 "2 S-III 7 . lxlO ""2 S-IV 7. .5xl0 "2 -methyl 1. .7xl0 "2 imidazole uncat (s -1 ) 1, . OxlO "5 Table II. The amino acid residues in the positions marked in bold text in the sequence below (SEQ. ID. No. 1) were altered for the peptides as shown below. 1 11 15 19 Ac-N-A-A-D-Nle-Ξ-A-X-I-K-H-L-A-X-X-Nle-A-X-X 20 23 -G-P-V-D 42 34 24 NH 2 -G-A-R-A-F-A-E-F-X-X-A-L-Q-E-A-Nle-Q-A-A Peptide Pos 8 Pos 14 Pos 15 Pos 18 Pos 19 Pos 33 Pos 34 S-I A E S A K K Orn (SEQ. ID. No. 2) S-II S E A A K K Orn (SEQ. ID. No. 3) S-III A K S A K K Orn (SEQ. ID. No. 4) S-IIIb A K S A K K A (SEQ. ID. No. 5) S-IIIc A K A A K K A (SEQ. ID. No. 6) S-IV S K A A K K Orn (SEQ. ID. No. 7) S-VIII A E S K s K A (SEQ. ID. No. 8) S-IX A E S K A K A (SEQ. ID. No. 9) s-x A E A A K K S (SEQ. ID. No. 10) S-XII S E A A K K A (SEQ. ID. No. 11) The sequences of these peptides are given in the appended sequence listing. As described above, if His(i) is flanked by Lys (i+4) then Lys (i+4) is exclusively amidated under the reaction conditions used here. If a His is flanked by a lysine residue in a neighboring helix in such a conformation that the Lys has a similar geometric relationship to the His, as has a Lys (i+4) or Lys (i-3) to a His(i), then this Lys will be amidated by the His in a direct acyla- tion reaction. In the sequences S-I (SEQ. ID. No. 2), S- II (SEQ. ID. No. 3), S-III (SEQ. ID. No. 4) and S-IV (SEQ. ID. No. 7) Orn-34 occupies such a position and in the absence of a Lys in position 15 Orn-34 will be preferentially amidated by His-11. When His-11 was flanked by Ser-15 As in S-I (SEQ. ID. No. 2), Figure 1, the degree of acylation of Orn-34 was enhanced considerably in comparison with that obtained with the sequence S-II (SEQ. ID. No. 3) , where the Ser was in position 8, showing that acylation is mediated by Ser-15. In these peptides no other Lys residues WERE in positions to accept acyl groups by direct transfer from His-11, and no amidations of residues other than Orn -34 were observed. When His-11 was flanked by Lys-14, in the peptide S-IV, Figure 1, in an i, i+3 configuration, no acylation of Lys-14 was found in agreement with the earlier report that i, i+3 configurations do not lead to amidation 5 . However, when the His-11 Lys-14 pair was supplemented by Ser-15, in the peptide S-III, amidation of Lys-14 was obtained, showing that Ser-15 mediated the transacylation reaction. Ser-15 can be conceived of as playing several roles in catalyzing the amidation of Lys- 14. However, the most likely is that it is acylated by the acylimidazole to form an, ester intermediate, that is subsequently trapped by Lys-14 in an intramolecular acyl- transfer reaction. Alternatively, Ser-15 can simply hydrogen bond to the developing oxyanion in the transition state of the amidation reaction or accept a hydrogen bond ω ω DO t → ι→ in o in o in o in rt μ- < Φ Φ X TJ l-x 9> 0 OJ rt μ- O Hi 03 l-x Φ 03 03 ι- χ X Φ ι- χ • < H σ> n o HI 03 Φ ! 0 φ Hi rt t- χ > Hl rt tr Φ ) ω DO t H> μ> in o in o in o in Ω rt et SD 03 <! S-ii Hi 3 SD μ- TJ Hi rt fi SD tr Ω hi CP Hi rt SD cp rt rt TJ S CD et Z 03 Z 0 Φ μ- 0 μ- 0J Φ Φ 0J <! 03 X Φ Φ Φ Ω I- 1 Φ ii 0 O P μ- tr fi o 0J Φ X PJ φ Φ 3 ti rt i rr t- χ rt fi CQ SD O ϋ μ- 9> et Φ 0J O i-x OJ HS Φ 0J CQ rt hj ø • -<. ii t- χ TS 3 μ- Φ ø φ φ ø μ- P CQ fi P Ω μ- Hi 0J Ω ø X t-x μ- ø 03 tr 1 Ul < h- 1 φ μ- O tr Φ fi ø μ- l→ O Ti φ 03 et 0 SD <! rt TS ■ ! ■ -<. • * <; ø TJ Ul μ- z CD Φ rt ø P • μ- 03 3 et rt SD rt tr Pi I- 1 - 0 03 SD μ- 03 03 N φ 0 Hi tr OJ Φ tr Pi SD H Φ (ϋ μ- ω μ- ø tr X 0 OJ 0 rt cp O 3 Φ CQ rt Φ t- χ ii Φ X 03 Hi 03 Pi P et Φ ø Ul H fi Φ ii 0 Hi 0 Φ Hi Ω OJ OJ Pi Φ fi Φ CQ Φ P X Ω et φ Φ Φ 0J <! Ul 3 0J T) ø μ- P . SD Hi Pi tr" rt ■ ξ tr Φ 0 Ω a P. rt fi I- 1 Φ Ω μ- 0 03 P Ti - Hi rt fi hi ø 3 φ ■ - tr μ- 0 Hi Φ O 03 Φ OJ ID ii rt SD et CQ Hi 0 Φ H μ- Φ h3 Φ Pi P. μ- 3 03 Φ rt Hi hi rt O tr Hi μ- rt rt X Hi X fi CQ tr tr X TJ J SD X ) φ ( P O 1 X et φ fi Hi Ω Pi t- χ Φ μ- TJ 0J Φ SD Ti CP Φ Φ φ fi t-x Ω Φ O CD 3 ti ø 1 TJ tr SD SD 0 tr μ- φ t-x- 0 fi rt tr rt φ Φ rt O rt fi Φ 0 SD Ul Φ μ- Φ Ω Ω l→ Φ rt 0i rt 3 0 P O φ 3 μ- TJ 03 rt H z rt <! 0 μ- Ω rt ϋ P rt et TS ø *<; rt l-x SD tr μ- CQ Ω μ- Hi • rt TJ O O rt rt fi Hi φ hi μ- Hi O O Φ 1 CQ μ- fi SD rt et 03 ι- χ μ- Φ Ω Φ HS tr rt Φ Φ 0 03 P μ- 03 SD φ SD Pi HI 3 !θi t *1 rt O OJ Hi μ- ii et <l HT 03 ii SD μ- rt μ- ti l et Pi Hi SD P HS μ- rt TJ ^ fi ts rt Pi iOi OJ ii CQ H- hi OJ μ- P fi P tr Φ rt Φ et 03 i 03 P ; X t- χ Φ 03 SD μ- Φ 3 0 hi rt tr CQ Φ Ω rt HS CP 0J OJ Pi Φ O 03 X Hi SOi 0 Hi CQ fi 9 φ SD rt 0 O Φ 0 Ω 0 • * • μ- t-x SD Φ Hi 03 TJ Hi 9> Φ μ- rt Φ 0 Ul X ii TJ Φ Hi Hi Hi CD l→ et 0 rt 0J 0 μ- 03 ø 03 et Hi 0 0J et fi rt HT fi SD 03 μ- I- 1 SD P. Hi 1 Φ 0 TS 0 et Φ rt O 03 tr OJ ii 0 μ- rt P fi μ- Φ iOi μ- O rt μ- rt OJ 0J μ- |x! Ω ti 03 Hi X . μ- tr Hi 0 Ω Hi TS Ω tr Pi SD Φ O fi TJ Pi Hi P - * <. φ X μ- Ω Ω H 0 Φ Φ <; φ Ω et *** 0J X TJ μ- φ Ω i ø 0 ) μ- Φ I-" H Z ø ^ ϋ. μ- H l→ tr rt H φ SD I- 1 l→ Ω 0 O φ fi Φ ^ fi rt rt 03 ^ 0J 0J t→ μ- φ OJ 0 φ tr T X SD Ω φ et SD Φ 03 03 ø 03 l→ ti Φ X μ- ø ^ 3 03 Hi CD ti H rt Φ ? •** . Φ CQ 3 ^ 9 X rt 03 TS rt et 03 rt Φ rt 03 Z O tr ϋ tr 03 O CQ . — . rt Φ z OJ Φ μ- ti Φ μ- X rt • μ- Φ t-x- 3 tr μ- 0J P rt Φ s=: - Pi fi μ- fi i- * J rt φ I- 1 fi O Pi SD ι- χ O rt o fi rt fi 0 SD Φ P-i SD X CQ 03 rt Φ O • — ' SD i -J Ω tr φ Φ < Φ 3 SD rt SJi P 0 ϋ SD H Φ 03 et μ- ø 03 t-x H 0 μ- μ- I- 1 £ •« 3 oo •** Φ P SD 91 OJ φ r μ- Φ ^ ø rt 03 Φ P Pi Φ Φ ø CQ O P TS μ- t-x <! t-x Ω rt Φ O Pi CP Ω rt t-x l Ω fi 03 Φ 03 Hi T5 O Hi rt TJ 03 CD SJi tr TJ Pi μ- ø rt ii P. ø Φ H SD X i—> SD Z SD 3 rt hi SD 0 et μ- X 0J hi Φ SD OJ 3 fi μ- ts Φ μ- μ- TJ Φ 0 Φ CQ OJ l→ X P Φ rt Φ SD O rt μ- O Hi Φ Pi O O fi rt 03 μ- 0 l-h CQ 03 < Φ μ- Ul Φ Φ 0 fi rt l Φ Pi O ti ι- χ 3 X Hi μ- ti P. rt < ^ - ^ μ- CQ rt Hi t-x- 03 Hi ø ø O Hi 0 μ- O ti Φ μ- 3 l Z Φ tr Ω μ- Φ Φ μ- O tr fi Φ Φ CQ 0 et CQ i O rt μ- 0 0 Φ CQ 0i tr μ- O 0r OJ 03 Φ 0J TJ rt μ- fi 1 0 φ SD μ- ti ii Hi 0J CQ Φ Hi Hi TS P 03 Ω rt φ Hi μ- < Ω Hi 0J μ- ø ω φ rt 0 Φ 0 Pi Φ ø tr 0J 03 SD ø rt SD Φ SD X 03 Ul o Ω rt O l t→ 0 HI μ- 0 £ rt 0 μ- 03 03 et OJ Φ h- 1 t- χ CQ tr TJ 03 P. rt OJ Ω 3 φ tr P O •- Hi 0 et TS X Hi 03 rt rt 3 Ω l-x CQ Φ φ φ μ- <! rt X *< Φ z ø 0 μ- 0 tn H Φ rt Φ μ- O μ- 3 03 OJ • Φ μ- 3 O Φ O J Ul 0 9i rt l CQ X X πs > ~< l H rt 0J TJ Hi P Pi φ μ- Hi 03 X 0 πs 0 Hi 03 et ø Hi > O μ- *< > Ω SD Pi μ- tr W tr Hi Z CQ ID SD rt 3 rt ■ - 0 P Hi CD φ ø Ul ø Ul l 0 0 ii rt TJ 03 O O Φ P μ- fi rt rt CP μ- . — . 1 fi 0 Φ ø μ- φ Ω μ- rt rt 03 φ tr o 03 Pi Ω 03 φ 03 rt tr μ- 0 φ P. fi I- 1 ii et Hi rt ii o ø 0J tr 0J tr 03 μ- -J TJ ι- χ 03 TJ CQ Φ HT φ O fi 0 μ- rt 0 φ O fi rt Φ < μ- X φ td rt 1 O SD ^ rt hi fi 0J Hi Φ Hi rt Hi tr t-x 0 rt SD Z tr 03 Φ 03 Φ μ- 0J ^ Z fi 03 μ- tr Φ o Hi rt fi Pi ti • SD * - TJ Φ rt X Φ Hi tr t→ m 03 HI — ' OJ OJ Hi μ- Q Φ l et TJ 0J Φ 0 TJ 0J rt 03 tr O Φ μ- Φ hi Φ OJ Φ SD Ω μ- 1 Ω 03 rt fi 03 Φ f . 03 SOi t- χ Hi SD fi Hi TJ μ- μ- ι- χ Pi Ω P O Φ P fi ι- χ 03 CD Φ TJ Φ OJ Pi TS i μ- SD tr μ- 0i 03 0 03 fi rt 03 t- χ < tr 3 Hi Pi • -< φ 1 Φ 03 SD O Pi Ω SD Φ SD 0 P μ- Hi rt Φ rt 1 0 tr φ et SD SD X ti rt tr -* 1 1 l-x 1 hi φ o 1 μ- SD μ- Φ 1 μ- hi 03 I- 1 - H CD 0J 03 1 fi < tr tr 1 03 1 03 Hi < Ω rt μ- 03 Φ Ω tr Φ fi Ul SD 1 SD 0 o Φ X Hl 1 Φ fi 1 • 03 03 tion of the reactants is clearly lost, although a very small one. For organic chemists the discovered reaction provides the opportunity to use a simple one-step reaction in aqueous solution to form new proteins with tailor made properties. The introduction of several ligands in a controlled way that recognize and bind known or unknown proteins can prove to be an important one in the upcoming era of proteomics . References : I. Baltzer, L., et al . , J. Chem. Soc . Perkin Trans. 2, 1996, 1671-1676. 2. Broo, K. , et al . , J. Am. Chem. Soc, 1996, 118, 8172- 8173. 3. Bruice, T. C. et al . , J. Am. Chem. Soc. 1958, 80, 2265-2271. 4. Lundh, A.-C, et al . , J. Chem. Soc. Perkin Trans. 2, 1997, 209-212. 5. Broo, K. , et al . , J, Chem. Soc. Perkin Trans. 2, 1997, 397-398. 6. Olofsson, S., et al . , J. Chem. Soc. Perkin Trans. 2, 1995, 2047-2056. 7. Olofsson, S., et al . , Folding Des . 1996, 1, 347-356. 8. Andersson, L., et al . , J. Org. Chem. 1998, 63, 1366- 1367. 9. Structure and mechanism in protein science, Alan Fersht, 1999, W.H. Freeman and Co Ch. 11. 10. Broo, K. S., et al . , J. Am. Chem. Soc. 1998, 120, 4063-4068. II. Nilsson, J. et al . Chem. Eur. J. 2000, 6, 2214-2220.

Claims: CLAIMS 1. A method for site-selective acyl transfer in or amidation of folded polypeptides and proteins based on the use of a chemical structure element comprising an imidazole function in position i flanked by at least one functional group to be amidated in position i+3+4k, where k is an integer equal to or higher than -1 or in position i -4 -4n, wherein n is an integer equal to or higher than 0, characterized in that it also comprises at least one activating group in position i+4+4n or i -3-4n, respectively, wherein n is as above. 2. A method according to claim 1, wherein k is -1, 0, 1 and n is 0, 1 or 2. 3. A method according to claim 1 or 2 , wherein said imidazole function is histidine. 4. A method according to any one of the claims 1 - 3, wherein said at least one functional group is lysine, ornithine or a diaminobutyric acid residue. 5. A method according to any one of the claims 1 - 4, wherein said at least one activating group is serine, threonine, tyrosine or cysteine. 6. A method according to any one of the claims 1 - 5, wherein said chemical structure element is a peptide. 7. A method according to claim 6, wherein said peptide has any of SEQ. ID. Nos . 1 - 11 given in the appended sequence listing. 8. A method according to claim 6 or 7 , wherein said peptide has SEQ. ID. No. 2, 4, 5, 8, 10 or 11 given in the appended sequence listing. 9. Use of the method according to any one of claims 1 - 8 for protecting proteins from proteolytic degradation. 10. Use of the method according to any one of claims 1 -8 for affecting protein folding. 11. Use of the method according to any one of claims 1 - 8 for vaccine development . 12. Use of the method according to any one of claims 1 - 8 to mimic naturally occurring components of the - immune system. 13. Use of the method according to any one of claims 1 - 8 to construct antagonists for components of the immune system. 14. Use of the method according to any one of claims 1 - 8 to construct agonists for components of the immune system. 15. Use of the method according to any one of claims 1 - 8 for production of antibodies . 16. Use of the method according to any one of claims 1 - 8 to incorporate, by amidation, into said chemical structure element one or more groups to form an artifi- cial receptor. 17. Use according to claim 16, wherein said artificial receptor is a protein chip for the identification of proteins from a cell lysate or in a solution. 18. Use according to claim 16, wherein said artifi- cial receptor is intended for use in separation or purification. 19. Use of the method according to any one of claims 1 - 8 for incorporation of a fluorescent group for identification of proteins. 20. Use of the method according to any one of claims 1 - 8 for incorporation of a paramagnetic relaxation agent for use in magnetic resonance imaging.

Inventors: BALTZER LARS, BALTZER, LARS
Applicants: A & SCIENCE INVEST AB, BALTZER LARS, A+ SCIENCE INVEST AB, BALTZER, LARS
Application year: 2001
Main classification: C07K
IPC codes: A61K49, C07K1, C07K14