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

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Effects of Fluorination on the Bioorganic Properties of Methionine 451

media. This approach, which is frequently utilized for the bioincorporation of other ﬂ uo-

rinated amino acids such as those of tryptophan, phenylalanine, and tyrosine, is applicable

to the introduction of DFM and TFM into almost any protein whose gene has been isolated

and whose expression can be controlled [9] . In the case of the DFM and TFM analogues,

placing the gene in an E. coli methionine auxotroph such as B834( λ DE3) reduces competi-

tion from the presence of intracellular biosynthesized methionine [15] . The incorporation

levels of DFM into proteins are higher than those for TFM even though the analogues

differ by only a single ﬂ uorine atom [25] . For example, for bacteriophage lambda lyso-

zyme, which contains three methionine residues (Met1, Met14, and Met107), incorpora-

tion levels for DFM approach wild - type levels ( > 20 mg/L of cell growth) with close to

100% incorporation (all three methionine residues replaced by DFM). TFM incorporation,

however, ranges from 31% TFM - labeled protein ( ∼ 15 mg/L) to 70% incorporation (2 mg/

L) with the requirement that small amounts of methionine be added to the growth media

to reduce TFM toxicity to E. coli cells (DFM was not found to be toxic to E. coli under

these bioincorporation experiments) [15] . The toxicity of TFM compared with DFM, an

analogue having only one less ﬂ uorine, is quite interesting but as yet the underlying

molecular details for the toxicity have not been elucidated, although their differential

processing by the enzyme methionine aminopeptidase (MAP) could be a contributing

factor (see below).

17.4

F NMR Spectroscopy of Proteins Incorporating DFM and TFM

F NMR spectroscopy is an extremely useful technique for the study of protein dynamics

and structure. The presence of the ﬂ uorine nucleus in these proteins due to the presence

of DFM or TFM enables the use of

F NMR to study their behavior in the presence of

ligands and inhibitors. Since there is no natural background of ﬂ uorine in proteins and as

the sensitivity of the

F nucleus to NMR detection approaches that of

H (83%), DFM/

TFM - labeled proteins can readily be investigated through this NMR method and several

interesting observations have already been made [7, 8] . TFM - labeled bacteriophage lyso-

zyme, because of partial TFM incorporation, is composed of an ensemble of partially

labeled proteins that are not readily separable by chromatographic techniques. Four distinct

F resonances (two of which integrate together to one TFM residue) are detected for this

ensemble, although only three methionines are present in the protein. Based on a series

of site - directed mutagenesis studies (M14L and M107L mutants labeled with TFM) as

well as use of the paramagnetic NMR line - broadening agent gadolinium(III) ethylenedi-

aminetetraacetic acid (Gd(III)EDTA), which affects surface exposed TFM residues, the

four

F NMR resonances were assigned to speciﬁ c residues in the protein [31] . Two

resonances occurring at − 39.32 ppm and − 39.82 ppm were assigned to Met1 and Met14,

respectively. However, Met107 was found to occur at either − 39.99 ppm or − 40.11 ppm

depending upon the extent of TFM incorporation. It was concluded that the position of

the

F resonance for TFM107 was dependent upon whether TFM was present at position

14 (producing the − 40.11 ppm resonance) or not (resulting in the − 39.99 ppm resonance

for TFM107). This implied that the presence of the larger TFM residue at position

14 compared with the smaller methionine residue somehow affects the environment

452 Fluorine in Medicinal Chemistry and Chemical Biology

surrounding position 107. When the x - ray structure of the bacteriophage lysozyme was

determined in collaboration with the group of Professor Albert Berghuis, it became clear

how this subtle change at position 14 might be transmitted to position 107 [32] . As shown

in Figure 17.3 , the protein was crystallized in complex with the hexasaccharide (GlcNAc)

an oligosaccharide that is not hydrolyzed by this bacteriophage lysozyme. The positions

of the three methionines are also shown in Figure 17.3 . Figure 17.4 presents a view of the

intervening region between Met14 and Met107. As can be seen from the structure, Met14

is in the hydrophobic core of the protein and tightly surrounded by several side - chains

(Ile113, Ile117, Leu142, and Phe146) that appear to contact the thiomethyl group of Met14.

Figure 17.3 Cartoon stereo diagram of the x - ray structure of wild - type bacteriophage lambda

lysozyme as solved for the (GlcNAc)

complex and showing the position of the three

methionine residues in the enzyme (PDB 1d9u) [32] . See color plate 17.3.

Figure 17.4 Close - up of the detailed interactions between Met14 and adjacent residues and

the location of Met107 (PDB 1d9u) [32] . See color plate 17.4.

Effects of Fluorination on the Bioorganic Properties of Methionine 453

As the size of the thiomethyl group is increased when TFM is introduced at this position,

the proximal residues would need to accommodate the increased steric demand produced

by the three ﬂ uorine atoms. Ile113 and Ile117 are positioned on a helix that contains

Arg110, Asp112, Gln115, and Arg119, and interestingly these residues surround Met107.

It is possible that the alteration of the

F resonance of TFM107 occurs due to the steric

interaction between TFM14 and proximal residues, and that these interactions are transmit-

ted through the helix to eventually result in a slightly altered environment for TFM107.

These observations indicate that TFM can be a very subtle probe of protein environment

and that the triﬂ uoromethyl moiety does slightly perturb protein structure, although not

sufﬁ ciently to alter enzymatic activity. In this case, bacteriophage lambda lysozyme enzy-

matic activity was measured by turbidimetric assay using chloroform - treated Escherichia

coli cells that lyse upon reaction with the lysozyme. Unfortunately, standard chromophore -

labeled oligosaccharides such as p - nitrophenol - linked saccharides, used extensively in the

study of hen egg white lysozyme, are not substrates for the phage enzyme.

Another interesting observation resulted when DFM was incorporated into the bac-

teriophage lysozyme. As mentioned previously, almost 100% incorporation of DFM into

the lysozyme occurs, which should simplify the

F spectrum as no protein ensemble exists

to complicate this spectrum. However, the

F NMR spectrum of the DFM - labeled lyso-

zyme exhibited the expected resonances for DFM1 ( − 91.2 ppm; doublet) and DFM107

( − 92.5 ppm; broader doublet) but a complex set of resonances (two quartets centered at

− 94.2 ppm and − 95.5 ppm) for the

F resonances associated with DFM14 (assignments

were made by site - directed mutagenesis, paramagnetic line broadening experiments, and

two - dimensional

F –

F COSY [correlation spectroscopy] NMR experiments) [25] . The

explanation for the NMR complexity of the

F signals for DFM14 is due to the fact that

the two ﬂ uorine atoms in DFM are diasterotopic due to the chirality of the amino acid,

and hence these two ﬂ uorine atoms are chemical shift inequivalent. As indicated above,

the tightly packed environment surrounding the DFM14 position will likely slow the side -

chain rotation of DFM14 compared with DFM at positions 1 and 107. This situation would

increase the observed chemical shift inequivalence of the two ﬂ uorines on DFM14, result-

ing in the two sets of quartets in the proton - coupled

F NMR spectrum (in addition to the

chemical shift difference, there would also be present both

F –

F and

H –

F coupling).

This effect is also observed in the study of the temperature effects on chemical shifts for

DFM itself, with low temperatures increasing the complexity of the

F resonances of the

diﬂ uoromethyl group. The DFM - labeled bacteriophage lambda lysozyme

F spectrum is

a stunning example of NMR chemical shift inequivalence.

17.5 DFM and TFM : Interactions with Platinum and

as Probes of Metalloenzymes

Although DFM and TFM are important contributions to the range of spectroscopic probes

available for application to biochemical studies, other properties of these ﬂ uorinated amino

acid analogues can also be of use. For example, the subtle change in size of the thiomethyl

group and the alteration in the electronic properties of the sulfur atom in these methionine

analogues could be useful properties to explore biological systems. In order to obtain

454 Fluorine in Medicinal Chemistry and Chemical Biology

additional fundamental information on these analogues, their interaction with metal com-

plexes was undertaken. For example, the reaction of methionine with K

PtCl

to produce

a methionine – platinum complex is well known (Figure 17.5 ) [33] . In addition, the rate of

sulfur inversion when complexed to platinum has been measured previously (Figure 17.6 )

[34, 35] . It was therefore of interest to determine the effect, if any, of ﬂ uorination on the

rate of complex formation as well as on the rate and energy barriers to sulfur inversion.

A preliminary comparison of the rate at which a series of methionine analogues react with

PtCl

resulted in the following values (in mM s

−

): selenomethionine 129 ± 4; methio-

nine 32 ± 2; ethionine 26.1 ± 0.9; DFM 0.50 ± 0.02; TFM 0.18 ± 0.04; norleucine

0.053 ± 0.001; control 0.021 ± 0.001 [36] . These observations signify that increasing ﬂ uo-

rination reduces the rate of interaction with the platinum metal center and that TFM is

especially slow in this regard. Nevertheless, reaction of DFM and TFM with K

PtCl

did

result in the formation of the corresponding platinum complexes, which were then studied

by NMR spectroscopy to determine the effect of ﬂ uorination on the rate of inversion of

the sulfur center [36] . The x - ray structure of the methionine – platinum complex is shown

in Figure 17.7 for reference [37] . Detailed dynamic NMR studies indicated that, within

experimental error, ﬂ uorination does not affect the rate of the sulfur inversion process in

these complexes as the inversion barriers for the DFM – and TFM – platinum complexes

were determined to be 16.4 ± 0.2 and 18 ± 1 kcal/mol, respectively, compared with a value

of 17 ± 1 kcal/mol for the methionine complex. The similarity of the inversion barriers

was also predicted from detailed computational studies at the B3LYP/SDD level.

Another aspect of the application of DFM and TFM analogues in protein engineering

is their relatively subtle steric changes compared to methionine. This alteration would be

Figure 17.5 Reaction of methionine and analogues with K

PtCl

Figure 17.6 Sulfur inversion process in methionine – platinum complexes.

Effects of Fluorination on the Bioorganic Properties of Methionine 455

difﬁ cult to accomplish by site - directed mutagenesis using only the standard 20 amino

acids. An example of the use of this characteristic is in the study of the key active site

methionine (Met214) in the P. aeruginosa alkaline protease (Figure 17.8 ) [38] . This

enzyme is a member of the metzincin family of zinc metalloproteases, which contain an

invariant methionine residue in their active site [39 – 41] . There are over 700 protein

members in this class. The role that the invariant methionine plays in this class of protease

is still uncertain, although a contribution to catalytic activity and/or protein stability for

some of the metzincins may occur [42, 43] . It was of interest to explore the capability of

DFM to substitute for this invariant methionine in the active site of the alkaline protease.

Successful incorporation of DFM could then be applied to the use of

F NMR spectros-

copy to investigate this class of protease. The invariant methionine residue is in close

proximity to the metal ligands of the protein (His176 and His186) (Figure 17.9 ), and

replacement of this residue by ﬂ uorinated methionines should also be interesting from a

protein structural perspective. Bioincorporation of DFM into the Met1 and Met214 posi-

tions of the protein was successfully accomplished after protein expression was optimized

in E. coli [27] . The presence of DFM in the invariant 214 position was found to be well

tolerated by the enzyme. Enzyme activity, as measured by hydrolysis of the artiﬁ cial sub-

strate Z - ArgArg - p - nitroanalide, was found to be comparable to that of the wild - type

enzyme [44] . Protein stability, as measured by differential scanning calorimetry, was also

found to be relatively unaffected by the presence of DFM. An interesting observation was

that the cellular removal of the N - terminal methionine from the alkaline protease by the

enzyme methionine aminopeptidase (MAP) was reduced when DFM replaced methionine.

It has been reported that the presence of TFM at the N - terminal of the green ﬂ uorescent

protein also affects normal N - terminal methionine processing [30] .

Recently DFM, TFM and other methionine analogues were utilized to explore the

contribution that methionine makes to the reduction - oxidation potential of the P. aerugi-

nosa metalloprotein azurin [26, 45] . Azurin is a copper metalloprotein that is involved in

Figure 17.7 Crystal structure of the methionine – PtCl

complex (coordinates from Wilson et

al. [37] ). See color plate 17.7.

456 Fluorine in Medicinal Chemistry and Chemical Biology

Figure 17.8 Ribbon diagram of the P. aeruginosa alkaline protease showing the active site

and the invariant methionine residue (PDB 1kap. [38] ). See color plate 17.8.

cellular redox reactions. In P. aeruginosa azurin, the active - site metal is ligated by His46,

His117, Cys112, the peptide amide of Gly45 and a more distal interaction with Met121

(Figure 17.10 ) [46] . There has been much speculation as to the contribution that Met makes

to the redox potential of this protein. The replacement of Met121 with a variety of non-

natural methionine analogues was undertaken to probe this interaction. In order to incor-

porate these analogues into the recombinant protein, expressed protein ligation was

employed [45] . Peptides (17 - mer) corresponding to residues 112 – 128 (H

N - CysThrPhePro

GlyHisSerAlaLeuMet * LysGlyThrLeuThrLeuLys - COOH) and containing the methionine

analogues were synthetically prepared by standard peptide synthesis and were linked to

recombinant C - terminal thioester activated recombinant azurin corresponding to residues

1 – 111 [26] . This methodology allowed for the semisynthetic preparation of azurin mutants

and is a convenient method to prepare site - selected modiﬁ cations containing unnatural

Effects of Fluorination on the Bioorganic Properties of Methionine 457

Figure 17.9 Close - up of the active - site region of P. aeruginosa alkaline protease showing

and protein ligands and the position of the thiomethyl group in close proximity to these

side - chains (PDB 1kap. [38] ). See color plate 17.9.

Figure 17.10 (a) Stereo view of the active - site structure of P. aeruginosa azurin (PDB 4azu)

[46] . (b) Tube diagram of the P. aeruginosa azurin showing the section of protein that was

contributed by intein ligation. See color plate 17.10.

458 Fluorine in Medicinal Chemistry and Chemical Biology

amino acids, which standard bioincorporation methods do not achieve. There was very

little structural alteration due to the presence of the methionine analogues as determined

by analysis of the S(Cys) - Cu charge - transfer bands in the electronic spectra as well as the

copper hyperﬁ ne coupling constants in the X - band electron paramagnetic resonance

spectra. Azurin containing methionine at position 121 had a measured redox potential of

341 ± 3 mV (vs. NHE), whereas DFM - and TFM - substituted azurin had redox potential

values of 329 ± 5 mV and 379 ± 4 mV, respectively. It was found through this approach

that there appears to be a linear relationship between the redox potential of the mutant

azurins and hydrophobicity of the amino acid analogues, a property difﬁ cult to identify

without the use of unnatural methionine analogues with varying properties. The application

of DFM and TFM to other metalloenzymes would be of interest both from a fundamental

perspective as well as in the engineering of altered electronic characteristics for other redox

proteins.

17.6 Structural Studies of DFM / TFM – Enzyme Complexes

Unfortunately, there is no x - ray structure of a DFM - or TFM - substituted protein available

to allow detailed study of ﬂ uorine – protein interactions in spite of efforts to accomplish

this (unpublished results). However, a few molecular structures have been determined for

DFM and TFM bound as substrates/inhibitors to two key enzymes involved in methionine

biochemistry, E. coli Met - tRNA synthetase and E. coli methionine aminopeptidase. Met -

tRNA synthetase is responsible for the ATP - dependent coupling of methionine on to its

cognate tRNA [47] . It would be the enzyme quintessential for the bioincorporation of

DFM/TFM into proteins. Insight into the preference of Met - tRNA synthetase for DFM

compared to TFM was obtained when the x - ray structures of the E. coli Met - tRNA syn-

thetase co - crystallized with DFM and with TFM were compared to that found with

methionine [48] . DFM was observed to bind in the same fashion as methionine, with

several key residues interacting in a similar fashion in the complexed active site (Figure

17.11 ). Both Met and DFM binding results in the movement of Tyr15, Trp253, and Phe300

toward the amino acid. This results in additional movement of other residues such as

Trp229 and Phe304 and stacking of Phe300 and Trp253, which is shown in Figure 17.11 a.

However the structure of the TFM - tRNA synthetase complex exhibited substantial differ-

ences (Figure 17.11 b) and no stacking interaction between Phe300 and Trp253 is present

in this complex. This difference alters the binding pocket shape and size for Met/DFM

compared to TFM. This may explain the lower bioincorporation levels of TFM compared

to DFM and, in addition, these x - ray structures provide critical insight into the enzymatic

process itself.

A report has also appeared detailing the molecular interactions between TFM and the

enzyme E. coli methionine aminopeptidase (MAP) [49] . The structure of MAP with bound

TFM shows some of the detailed interactions that exist between this ﬂ uorinated amino

acid and the enzyme and its interaction with the hydrophobic pocket composed of Cys59,

Tyr62, Tyr65, Cys70, His79, Phe177, and Trp221 (Figure 17.12 ). Overall the structure

of the TFM complex appears similar to that of the Met complex. Further analyses are

required to understand the factors that contribute to the apparently lower processing rates

Effects of Fluorination on the Bioorganic Properties of Methionine 459

Figure 17.11 X - ray structures of (a) DFM (PDB 1pfv) [48] and (b) TFM (PDB 1pfw) [48]

complexes with Met - tRNA synthetase from E. coli . See color plate 17.11.

Figure 17.12 Stereo view of the active - site interactions of TFM with E. coli methionine

aminopeptidase (PDB 1c22) [49] . See color plate 17.12.

460 Fluorine in Medicinal Chemistry and Chemical Biology

of ﬂ uorinated N - terminal residues. Nevertheless these structures contribute additional

insight into the molecular recognition between ﬂ uorinated amino acids and proteins.

In conclusion, the application of DFM and TFM to bioorganic chemistry has led to

new information on how ﬂ uorinated compounds interact with enzymes, has provided novel

F NMR biophysical probes for use in biochemistry, and has led to subtle structurally and

electronically modiﬁ ed analogues of methionine with which to probe enzymes. The addi-

tion of several ﬂ uorine atoms to a simple structure such as methionine has certainly led

to an interesting series of adventures in bioorganic chemistry.

Acknowledgments

The author gratefully acknowledges the past and present contributions of his undergradu-

ate and graduate students, research associates, and collaborators. Funding from NSERC

(Canada) and the University of Waterloo is also gratefully acknowledged.

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