Using the Potential of Fluorine for Peptide and Protein Modification

Chapter 35

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Using the Potential of Fluorine for Peptide and Protein Modification Christian Jäckel and Beate Koksch* Freie Universität Berlin, Institut für Chemie, Takustrasse 3, 14195 Berlin, Germany

Methods for the enzyme-catalyzed resolution of a broad variety of fluorinated amino acids as well as for their incorporation into peptides and proteins using commercially available proteases have been developed. The synthetic strategies described here extend the scope of methods available for site-specific peptide and protein modification by fluorinated amino acids using simple and environmentally attractive routes. Moreover, a new screening system which has been developed for a systematic investigation of the molecular interactions of fluoro-substituted amino acids with native polypeptides is introduced.

Extending the spectra of building blocks which can be used for peptide and protein engineering beyond the natural amino acids broadens the scope of peptide and proteins. (7-5) Highly fimctionalized amino acid residues can serve as valuable tools to be used as biophysical probes for detailed studies of structure-function relationships or for the construction of tailor-made biomolecules which will expand the repertoire of protein functions. Many functional groups like e.g. halides are rarely found in the natural amino acid pool. Incorporation of the unique electronic properties of fluorine into amino acids creates, therefore, a new and exciting class of building blocks for peptide and protein modification. (4) Beta-fluorinated amino acids themselves have

© 2005 American Chemical Society

Soloshonok; Fluorine-Containing Synthons ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

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612 gained prominence as mechanism based inhibitors of amino acid decarboxylases and transaminases. (5) C -fluoroalkyl substituted amino acids bearing a fluorinated substituent instead of the α-proton are known to be able to increase metabolic stability (6) of peptides as well as to stabilize peptide secondary structure. (7) The incorporation of fluorine usually shows dramatic effects on protein stability, protein-protein interactions, and the physical properties of protein based materials. (8-11) Furthermore, the F atom as well as the C-F bond serves as a highly specific and powerful label for spectroscopic investigations of pathways, metabolisms, and structure-activity relationships using NMR or Raman spectroscopy, respectively. (12-14) The impact of the fluorine substitution on all of the above-mentioned peptide and protein properties strongly depends on the position as well as the content of fluorine substitution within a special amino acid while the main discrimination has to be made between side chainfluorinationand C -fluoroalkyl substitution. This difference not only dictates the properties exerted by the fluorinated building block but also the methods which have to be used for amino acid synthesis as well as for their incorporation into peptides. It is part of our research program to develop routine methods for the synthesis of a broad variety offluorinatedamino acids as well as for their incorporation into peptides and proteins. Moreover, we study the properties offluorinatedamino acids and their interaction pattern with native amino acids within a native polypeptide environment. Thefirstpart of this review, therefore, will summarize new methods for resolution of racemic C -fluoroalkyl substituted amino acids as well as the protease-catalyzed incorporation of these sterically demanding amino acids into peptides. The second part introduces a new screening system which has been developed for a systematic investigation of the molecular interactions offluoro-substitutedamino acids with native polypeptides. a


,9

a

a

a

Part 1. Methods for Enzymatic Resolution of Racemic C Fluoroalkyl Substituted Amino Acids and for their ProteaseCatalyzed Incorporation into Peptides a

1.1. Enzymatic Resolution of Racemic C -Fluoroalkyl Substituted Amino Acids a

Several routes towards racemic C -fluoroalkyl substituted amino acids in which the α-proton is substituted by a fluoroalkyl group have been developed. The


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most convenient synthesis of this class offluorinatedamino acids is based on an amidoalkylation of carbon nucleophiles with highly electrophilic acylimines of 3,3,3-trifluoropyruvate (5,15-22; Scheme 1). FC R HN^C0 Me

F

? 3

F (yC0 Me 3

^

2

3

^C0 Me 2

2

2

1

R 0"%

1

° Downloaded by UNIV OF CALIFORNIA SAN DIEGO on January 4, 2017 | http://pubs.acs.org Publication Date: July 21, 2005 | doi: 10.1021/bk-2005-0911.ch035

N

R cAo 1

2

R =Ζ

R = alkyl, benzyl

a. R OCONH , b. (CF CO) 0/pyridine, c. R MgX, d. H 0 1

2

2

2

3

+

3

a

Scheme 1. Synthesis route towards fully protected C -fluoroalkyl substituted amino acids.

This route enables the synthesis of a-(trifluoromethyl) amino acids (aTfm amino acids) with orthogonal protective groups. (23-26) Analogously, a(difluoromethyl) α-amino acids (aDfm amino acids), the virtually unknown a(chlorodifluoromethyl) and a-(bromodifluoromethyl) α-amino acids can be obtained via addition of C nucleophiles to acylimines of corresponding partially fluorinated pyruvates. (27) Due to an increasing interest in fluoroalkyl amino acids for peptide and protein modification and considering the divergent biological activities of the enantiomers of C -fluoroalkyl substituted amino acids and their diastereomeric peptide derivatives, the availability of these compounds in enantiomerically pure form is highly desirable. Important effords have been made in the development of new methods for the enantioseiective preparation of β-fluorinated α-amino acids during the last years. (28-30) However, several synthetic routes to optically pure C -fluoroalkyl substituted amino acids rely on chemical (31-33) and enzymatic resolution. (34) A promising strategy for a diastereoselective synthesis of aTfm amino acids proceeds via amidoalkylation of carbon nucleophiles with in situ formed homochiral cyclic acyl imines (35,36). The dioxopiperazines (DOP) obtained with good stereoselectivity can be transformed into homochiral dipeptide esters by regioselective acidolysis in methanol. (35) However, the majority of fluorinated C ' -dialkylated amino acids is prepared chemically followed by enzymatic resolution of the enantiomers. The separation of the optical isomers of H-(aTfm)Ala-OH by partial hydrolysis of the racemic N-trifluoroacetyl derivative with hog kidney aminoacylase has been reported by Keller et al. (34) It was reported by our group that proteases like subtilisin, achymotrypsin or papain accept C -fluoroalkyl substituted amino acid esters as substrates only to a very limited extent. (37) Therefore, the application of these a

a

a a

a


614

a

proteases for the resolution of enantiomeric C -fluoroalkyl substituted amino acid derivatives is excluded except for Z-(aTfm)Gly-OMe. (38) At DSM Pharma Chemicals (Geleen, The Netherlands) several methods for the preparation of enantiomerically pure C ' -dialkylated amino acids via enzymatic resolution of racemic amino acid amides have been developed. Amidases from Mycobacterium neoaurum (ATCC 25795) and Ochrobactrum anthropi (NCIMB 40321), both exhibiting a high L-stereoselectivity, have been applied for largescale preparation of many different optically active C^-dialkylated amino acids (39,40; Scheme 2). It was, therefore, interesting to study the influence of the electronically modified amino acid derivatives on individual enzyme-substrate interactions and, thus, the catalytic efficiency and enantioselectivity of the amidases (41; Table I).


a a

Scheme

a

2. Enzymatic resolution of racemic C -fluoroalkyl amino acid amides

Amidase from Mycobacterium neoaurum (ATCC 25795) hydrolyzes R,S-K(aTfm)Ala-NH , *,S-H-(aCF Cl)Ala-NH and *,S-H-(aCF Br)Ala-NH with high enantioselectivity (E > 200) to give the pure Λ-amino acids. In case of R,SH-(aTfm)Ala-NH the reaction was carried out in preparative scale and both the amino acid and the unconverted amide were isolated. As a proof of the enantioselectivity of the amidase reaction the amino acid was coupled to H-AlaN H via standard chemical peptide synthesis and the amino acid amide was likewise coupled to Z-Ala-OH. The resulting dipeptide products were shown to be diastereoisomerically pure by F NMR as well as H NMR analysis. Mycobacterium neoaurum usually expresses a high enantioselectivity for the Lform of a racemic mixture. The absolute configuration of the converted H(aTfm)Ala-NH could be assigned to be R which corresponds to D-Ala if comparing the positions of the methyl groups. Apparently, the Tfm group as the larger of the two substituents at the C -atom is bound by the enzyme in the pocket which usually binds the methyl group of Z-Ala. To what extent the electronic properties of the fluorine substituents add to the size effect would need to be investigated further. The growing size of the substituents at the halogenated group does not seem to influence the enantioselectivity of the enzyme (E > 200) for reaction with *,£-H-(aCF Cl)Ala-NH and (aCF Br)Ala-NH . Merely the reaction rate is significantly lower for these substrates than for the aTfm congener. 2

2

2

2

2

2

2

I9

!

2

a

2

2

2

2


615 Ochrobactrum anthropi (NCIMB 40321) accepts R,S-H-(aDfm)Phe-NH as substrate and allows, for the first time, a successful enantioselective enzymatic hydrolysis of a C -fluoroalkyl substituted Phe derivative. In contrast, Phe derivatives which bear more than two halogen residues at the C -alkyl group are not accepted by the two enzymes. No significant hydrolysis was observed in these cases. These results indicate that the steric constraint exhibited by a trifluoromethyl or difluorochloromethyl group combined with the presence of a benzyl group, both at the α-carbon atom of an amino acid, is too high even for enzymes which provide a wide substrate specificity such as Ochrobactrum anthropi amidase. However, this technology can now be applied for the preparation of a variety of enantiopure C -fluoroalkyl substituted amino acids in preparative scale. (41) 2

a


a

a

a

Table I. Enzymatic Resolutions of Racemic C -Fluoroalkyl Amino Acid Amides using Amidases from Mycobacterium Neoaurum and Ochrobactrum Anthropi 2

R

1

R

1 CF (Tfm) 2 CF C1 3

3

2

3

3 CF Br 2

6 CH Ph 2

ConversionAmide Amino Ε value" ee(%) Acid ee (%) (%) Mycobacterium 47 98.0 96.0 >200 CH neoaurum CH Mycobacterium 48 99.5 94.7 >200 neoaurum Mycobacterium 49 99.5 94.8 >200 CH neoaurum CF H Ochrobactrum 58 25 98.7 67.9 (Dfm) anthropi Enzyme

3

b

2

a)

Ε values were calculated on the basis of the experimentally determined; e.e. values of the amide and the acid. This assignment is tentative. b )

1.2, Protease Catalyzed Peptide Synthesis for the Site-Specific Incorporation of α-Fluoroalkyl Amino Acids into Peptides In our attempt to provide a broad variety of methods for the fast and simple incorporation of fluorinated amino acids into peptides, methods have been developed using commercially available proteases that make the direct enzymatic coupling of these sterically demanding and electronically modified amino acids possible. The synthetic strategy introduced here extends the scope of methods available for site-specific peptide and protein modification by fluorinated amino acids using simple and environmentally attractive routes.


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1.2.1. Incorporation of a-Fluoroalkyl Amino Acids into Peptides using Try and a-Chymotrypsin through Substrate and Medium Engineering Effective site-specific incorporation of a wide variety of nonnatural amino acids into peptides and proteins remains a topic of high interest as it provides the opportunity for a more detailed understanding of protein structure and function. The combination of chemical synthetic methods with enzymatic peptide bond formation for the site-specific incorporation of nonnatural amino acids into peptides and proteins represents an attractive alternative to classical peptide chemistry. Enzymes work generally racemization free, highly regio- and stereoselectively, under mild reaction conditions, and require only minimal side chain protection. (42,43) However, the substrate specificity of available proteases usually restricts the number of residues between which a peptide bond can be synthesized. While di- and tripeptide methyl esters containing JV-terminal aTfm amino acids are accepted as substrates by subtilisin, α-chymotrypsin, trypsin, and clostripain (37,44-46), direct enzymatic coupling of α-fluoroalkyl amino acids have been unsuccessful. Even in the case of Z-(aTfm)Gly-OMe which was shown to be a very specific substrate for subtilisin, protease-catalyzed peptide synthesis failed. (38) Recently, a powerful concept was established which overcomes these limitations of the classical enzymatic approach. (47-52) This concept is based on the binding site specific 4-guanidinophenyl ester (OGp) functionality to mediate acceptance of nonspecific amino acid moieties in the specificity-determining Sj position of the enzyme (notation according to Schechter and Berger; 53). Applying the advantages of the substrate mimetic concept to fluoroalkyl amino acids, we have succeeded for the first time in incorporating these sterically demanding C^-dialkyl amino acids into the Pi position of peptides enzymatically. (54) 4-Guanidinophenylester of C^-fluoroalkyl Ala derivatives or Aib, respectively, can be easily prepared by reaction of the N-protected amino acids with 4-[iV,iV"bis(tert.-butyloxycarbonyl)guanidino]phenol using TBTU as coupling reagent. The sterically higher demanding C^-fluoroalkyl or methyl Leu and Phe derivatives, respectively, can be synthesized in high yields as well, but have to be activated with DIC/HOAt and reacted with the lithium salt of the guanidinophenol (Scheme 3). aDfm and aTfm substituted Ala, Leu, and Phe derivatives can be coupled directly to various nucleophiles - different in sequence and length - by trypsin (see Table II). Remarkably, in all cases the efficiency of peptide synthesis is much higher for aDfm-substituted amino acids compared to aTfm- as well as amethyl-substituted derivatives. Remarkable differences in product yields between the (aDfm)Ala derivative and both Aib and (aTfm)Ala substrate


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mimetics indicate a significant influence of the second α-substituent at the acyl donor on individual enzyme-substrate interactions. The efficiency of peptide synthesis using Z-(aDfm)Ala-OGp as the acyl component is at least twice as high in most cases as for the corresponding Aib and (aTfm)Ala derivatives, respectively, while product yields for both of the latter were found within the same range.

Scheme 3. Synthesis of C* -dialkyl amino acid-4-guanidinophenyl esters. R : CH , CF H, CF ; R : CH C^Î CH CH(CH ) ; R : CH ; a: DIC, HOAt, THF; b: η-butyl lithium, THF; c: TBTU, DMA, DMF; d: TFA, ultra sound. a

1

2

3

2

3

3

2

Sf

2

3 2

3

Interpreting the differences in product yields between (aDfm)Ala derivatives and Aib and (aTfm)Ala substrate mimetics, respectively, the use of racemic mixtures in case of Z-protected (aDfm)Ala and (aTfm)Ala esters unlike for the Aib derivative has to be taken into account. A significant influence of the absolute configuration of the dialkyl amino acids on enzyme-substrate interactions within the active site of trypsin seems to be a more likely reason for this difference than the steric demand of the second substituent (alkyl or


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fluoroalkyl, respectively) at the C^-atom. To prove this assumption, enzymatic peptide synthesis of Z-(aDfm)Ala-Met-NH and Z-(aTfm)Ala-Met-NH was carried out in a semi-preparative scale. Diastereomers were separated by HPLC and characterized by F NMR. In case of the aTfm-substituted peptide a diastereomer-ratio of 1:3 was found, while for the corresponding aDfm substituted peptide the ratio was 1:1. Obviously, both enantiomers of the Z(aDfm)Ala-enzyme complexes are deacylated by the nucleophile at the same rate which gives identical product yields. In contrast, one enantiomer of the Z(aTfm)Ala-enzyme complex appears to be hydrolyzed faster by water than aminolyzed by the nucleophile, resulting in the formation of the amino acid instead of peptide bond formation. Moreover, the ability of the Dfm group to function as a hydrogen bond donor compared to the Tfm functionality certainly contributes to the observed difference in the interaction pattern between the fluoroalkyl amino acids and the enzyme. (55,56) Unlike the Tfm group, the aDfm amino acid could interact with the S' region of the enzyme stabilizing the acyl enzyme intermediate. This would lead to a delayed reaction of at least one of the enantiomers with any kind of nucleophile which would result in a simultaneous aminolysis. 2

2


l9

Table II. Yields (%) of Trypsin-Catalyzed Peptide Synthesis using Substrate Mimetics of C^-Dialkylated Amino Acids. Acyl donor

Z-X °Ala-OGp c

Acyl acceptor X

Using the Potential of Fluorine for Peptide and Protein Modification

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