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17

Effects of Fluorination on the

Bioorganic Properties of Methionine

John F. Honek

17.1 Introduction

Amino acids, peptides and proteins play essential roles in living systems. As chemists, we

have the ability to fabricate new structures of great complexity. Yet frequently the synthesis

and application of even the simplest chemical structures can contribute substantial infor-

mation on living systems. This has certainly been true for the application of ﬂ uorine

chemistry to the synthesis of ﬂ uorinated amino acids [1, 2] . These molecules are not only

interesting in their own right but may act as potent mechanism - based enzyme inhibitors

that may have application in medicine or diagnostics, or they can be valuable probes that,

incorporated into peptides or proteins, elucidate fundamental biological chemistry or

uncover new aspects of biochemical structure and function [3 – 5] .

Why ﬂ uorine? Its van der Waals radius is 1.47 Å compared with 1.2 Å for hydrogen

and 1.57 Å for oxygen and with small molecules such as amino acids, the introduction of

a large substituent would likely be completely disruptive to its biological function [6] .

However, a relatively small atom such as ﬂ uorine introduces a minor alteration in sterics

to the molecule. In addition, the presence of a ﬂ uorine atom introduces a powerful nuclear

magnetic resonance (NMR) probe (

19

F) into the molecule. Fluorinated analogues of tyro-

sine, phenylalanine, and tryptophan have been and continue to be utilized as biological

probes in biological chemistry and as important reporter groups in protein folding and

ligand binding studies [7 – 10] . In addition, the electronic perturbations that can occur upon

addition of the ﬂ uorine nucleus into a structure such as an amino acid can also be used to

Fluorine in Medicinal Chemistry and Chemical Biology Edited by Iwao Ojima

448 Fluorine in Medicinal Chemistry and Chemical Biology

evaluate the electronic aspects involved in molecular recognition. The synthetic prepara-

tion of ﬂ uorinated amino acids and their application in medicinal chemistry and biochem-

istry has contributed much to our fundamental understanding of molecular recognition as

well. Although it is easy to replace a hydrogen atom by a ﬂ uorine atom in a chemical

structure when drawing this structure, the chemistry involved in actually making the ﬂ uo-

rinated target molecule can usually be quite challenging. Frequently new synthetic

approaches may be necessary to accommodate either available ﬂ uorinated starting materi-

als or ﬂ uorinating reagents. Regardless of the synthetic difﬁ culties that might be encoun-

tered in the preparation of various ﬂ uorinated amino acids, the large impact that ﬂ uorine

can play by its presence continues to be a major reason for chemists and biochemists to

include the use of ﬂ uorinated amino acids in their research programs.

This chapter focuses on how ﬂ uorinated methionine analogues interact with biological

systems. Speciﬁ cally the compounds l - monoﬂ uoromethionine ( l - ( S ) - (monoﬂ uoromethyl)

homocysteine; MFM), l - diﬂ uoromethionine ( l - ( S ) - (diﬂ uoromethyl)homocysteine; DFM),

and l - triﬂ uoromethionine ( l - ( S ) - (triﬂ uoromethyl(homocysteine); TFM) will be the subject

of this article (Figure 17.1 ). A discussion of their syntheses and chemical and conforma-

tional properties will be presented. This will be followed by a discussion of their incorpora-

tion into peptides and proteins and the properties of the resulting ﬂ uorinated biomolecules.

The

19

F NMR spectroscopic characteristics of these biomolecules will be covered along

with how these resonances have revealed information on the properties of the diﬂ uoro - and

triﬂ uoromethyl moiety (and on the proteins as well!). Further elaboration of these amino

acids with metalloenzymes completes the survey.

17.2 Syntheses and Characteristics of Fluorinated Methionines

A number of methods for preparing protected and unprotected MFM, DFM, and TFM

have been reported. In the case of MFM, protected versions of MFM have been reported

utilizing either xenon diﬂ uoride on the sulﬁ de or diethylaminosulfur triﬂ uoride (DAST)

on the protected methionine sulfoxide [11, 12] . In the DAST approach, the N - acetylated

methyl ester of methionine sulfoxide was reacted with DAST and ZnI

2

(60 ° C, 12 h) to

produce the targeted protected monoﬂ uoromethyl sulﬁ de in 67% yield. Unfortunately, the

lack of stability of the monoﬂ uoromethyl sulﬁ de group to chromatography and to the

aqueous conditions required for deprotection prevented the isolation of deprotected MFM.

This is further experimental evidence for the lack of stability of an α - monoﬂ uoroalkyl

sulﬁ de group as aqueous instability has been observed for a nucleoside containing a similar

functional group [13] .

Figure 17.1 The chemical structures of L - methionine, and monoﬂ uoro - (MFM), diﬂ uoro -

(DFM) and triﬂ uoromethionine (TFM).

Effects of Fluorination on the Bioorganic Properties of Methionine 449

Dannley and Taborsky ﬁ rst reported the synthesis of racemic d , l - TFM utilizing

(CF

3

S)

2

Hg as the source of the triﬂ uoromethylthio group [14] . Unfortunately, low yields

were reported with this approach (11% in ﬁ ve chemical steps). Improved synthetic routes

have been reported and are dependent upon the photochemical reaction of various CF

3

-

containing reagents with homocysteine or N - acetylthiolactone [12, 15] . Reagents such as

triﬂ uoromethyl iodide or S - triﬂ uoromethyl diaryl sulfonium salts are able to transfer a tri-

ﬂ uoromethyl group to thiols, and these reagents have been useful in this regard [12, 15 – 17] .

Starting with inexpensive racemic N - acetylhomocysteine thiolactone, the racemic N -

acetyltriﬂ uoromethionine methyl ester was deprotected by base followed by enzymatic

resolution using acylase I. Similar approaches have been reported for the successful synthe-

sis of DFM, that is, by reaction of l - homocysteine or racemic N - acetylhomocysteine thio-

lactone under basic conditions with Freon - 22 (chlorodiﬂ uoromethane) [12, 18] . A summary

of several of the approaches to preparation of DFM and TFM is shown in Figure 17.2 .

The introduction of ﬂ uorine into the methyl group of methionine alters a

number of properties. For example, calculations have indicated that the overall

hydrophobicity would be altered upon ﬂ uorination of the thiomethyl group

log . ; log . ; log .PP P

CH SCH CF HSCH CF SCH

33 2 3 33

046 177 239===

()

[19] . Computational model-

ing of the side - chain of TFM indicates a shortening of the carbon – sulfur bond of CF

3

– S

and a lengthening of the S – CH

2

bond [20] . This results in a lower energy barrier for dihe-

dral angle rotation in the TFM side - chain, with dihedral angles of 80 ° , 180 ° , and 280 °

being lower - energy conformations. The energy barrier between these minima are on the

order of 0.8 kJ/mol compared with the methionine side - chain, which has a calculated

energy barrier on the order of 1.5 kJ/mol on the basis of relaxed potential energy scans at

the B3LYP/6 – 31+G(d,p) level [20] .

One of the goals for the study of methionine biochemistry utilizing these

ﬂ uorinated methionines was to control, in a stepwise fashion, the electron density and the

Figure 17.2 Summary of some of the synthetic routes to DFM and TFM.

450 Fluorine in Medicinal Chemistry and Chemical Biology

nucleophilicity of the thioether moiety by increasing ﬂ uorination adjacent to the sulfur

atom. With respect to the effect that ﬂ uorination makes on the steric properties of the

methyl group in methionine, it has been suggested that a triﬂ uoromethyl moiety may be

similar in size to the isopropyl group [6] . Computational calculations at the ab initio level

(RHF/6 – 31G * ) have resulted in the calculated volume enclosed by the electron density

surface computed at the 0.002 electrons/(atomic unit)

3

level to be 71.2 Å

3

, 80.9 Å

3

, and

84.4 Å

3

for the simple molecules CH

3

SCH

3

, CH

3

SCHF

2

, and CH

3

SCF

3

, respectively [19] .

Electronic properties of the thiomethyl function in methionine are also seen to change

upon ﬂ uorination. Calculations using density functional theory at B3LYP/6 – 31+G(d,p)//

B3LYP/6 – 31+G(d,p) levels have determined the Mulliken charges on the sulfur atoms of

CH

3

SCH

3

, CH

3

SCHF

2

, and CH

3

SCF

3

to be 0.079e, 0.139e, and 0.212e respectively and

the CHelpG charges [21] ﬁ tted to the electrostatic potential according to the CHelpG

scheme are − 0.286e, − 0.221e, and − 0.149e for the sulfur atoms in CH

3

SCH

3

, CH

3

SCHF

2

,

and CH

3

SCF

3

respectively (unpublished results). Hence, the introduction of ﬂ uorine into

the methionine structure will perturb the sterics as well as the electronic properties of the

side - chain. However, these alterations are made in a stepwise direction DFM → TFM

when both analogues are studied in parallel in a particular biological system.

17.3 Replacement of Methionine Residues in Peptides and

Proteins by DFM and TFM

To our knowledge, the only report of the application of DFM and TFM to peptides has

been on the effect of these replacements on the chemotactic activity of the tripeptide

formyl - Met - Leu - Phe [19] . The methionine residue in this peptide has been shown to be

critical to the bioactivity of the peptide, as replacement by other methionine analogues

such as ethionine, norleucine, S - methylcysteine, S - ethylcysteine or 2 - aminoheptanoic acid

results in ∼ 4 - fold to 264 - fold decrease in chemotactic activity in human and rabbit neu-

trophil migration assays [22 – 24] . Interestingly the DFM - and TFM - containing peptides

were found to enhance by ∼ 10 - fold the directed migration responses of human blood

neutrophils. Studies of the effects of DFM and TFM on the bioactivity of other peptides

would certainly be worthy of further investigation.

In contrast to the lack of application of DFM and TFM to peptide biochemistry, bio-

incorporation of these analogues into several proteins has been reported. DFM and TFM

have been successfully incorporated into recombinant bacteriophage lambda lysozyme

(DFM and TFM) [15, 25] , Pseudomonas aeruginosa azurin (DFM and TFM) [26] , P.

aeruginosa alkaline protease (DFM) [27] , calmodulin (DFM) [28] , Escherichia coli

leucine - isoleucine - valine (LIV) binding protein (DFM) [29] , and a mutant of the Aequorea

victoria green ﬂ uorescent protein (TFM) [30] . The presence of these ﬂ uorinated amino

acids in those proteins does not negatively affect either the enzymatic activity or the bio-

activity of the proteins, an important ingredient in the characteristics that an amino acid

analogue must have if it is to serve as a useful biophysical probe. The bioincorporation of

DFM or TFM into the above proteins (except for the P. aeruginosa azurin) has been readily

accomplished by induction of the target gene ’ s expression (induced by isopropyl - β - d -

thiogalactopyranoside, IPTG) in the presence of either DFM or TFM in minimal growth

Ojima I. (ed.) Fluorine in Medicinal Chemistry and Chemical Biology

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