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

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Applications of Fluorinated Amino

Acids and Peptides to Chemical

Biology and Pharmacology

Fluorine in Medicinal Chemistry and Chemical Biology Edited by Iwao Ojima

Application of Artiﬁ cial Model

Systems to Study the Interactions

of Fluorinated Amino Acids within

the Native Environment of

Coiled Coil Proteins

Mario Salwiczek , Toni Vagt , and Beate Koksch

15.1 Introduction

Because of the unique physicochemical properties of carbon - bound ﬂ uorine and due to

the fact that it does not appear within the pool of ribosomally encoded amino acids, ﬂ uo-

rinated analogues can be used as powerful analytical labels to investigate protein structure

and protein – ligand interactions [1] . Furthermore, ﬂ uorine ’ s impact on the structure, bio-

logical activity, and stability of polypeptides makes it an interesting substituent for protein

modiﬁ cation [2 – 5] as well as for the de novo design of artiﬁ cial proteins [6] . In this context,

ﬂ uorinated analogues of hydrophobic amino acids show great promise as modulators for

the stability and self - organization of protein folding motifs whose interactions are largely

based on complementary hydrophobic side - chain packing. Global substitution of apolar

residues by ﬂ uorinated analogues within the hydrophobic core of α - helical coiled coils

usually results in a strong thermodynamic stabilization and the speciﬁ c formation of ﬂ uo-

rous interfaces that strongly direct the self - sorting of these peptides [7] . In addition to such

“ teﬂ on ” proteins [8] per se, an interesting task is also to investigate how ﬂ uorinated amino

Fluorine in Medicinal Chemistry and Chemical Biology Edited by Iwao Ojima

392 Fluorine in Medicinal Chemistry and Chemical Biology

acids interact with their naturally occurring counterparts. However systematic approaches

toward this goal have been published only very recently [9] . Numerous attempts at inves-

tigating the interactions of comparably small nonpeptidic organic molecules with enzymes

indicate that, in addition to the hydrophobicity of ﬂ uoroalkyl groups, their polarity also

plays an important role [10 – 14] . Polar interactions of carbon bound ﬂ uorine were shown

to induce a favorable binding of a potential inhibitor to the thrombin active site [13] .

Moreover, ﬂ uorine - induced polarity may result in disadvantageous, inverse ﬂ uorous effects

such as a decrease in lipophilicity [10, 15] . Regarding polar interactions of ﬂ uorine, it is

also important to note that ﬂ uorine scientists have not yet fully agreed on whether carbon -

bound ﬂ uorine may accept hydrogen bonds, especially from the functional groups of pro-

teins [16] . Accordingly, to this day, there is no consistent opinion on ﬂ uorine ’ s behavior

as a nonnative “ functional group ” in amino acid side - chains. Such a speciﬁ cation, however,

would be the most important precondition for enabling a directed application of ﬂ uorine ’ s

unique properties in the engineering of peptides and proteins and their interactions with

one another.

Scientiﬁ c approaches that attempt to rationalize ﬂ uorine ’ s effects on the interaction

of polypeptides with native proteins usually rely on model systems with a precisely deﬁ ned

interaction pattern that mimics a native environment. With the objective of unraveling the

effects of even single ﬂ uorine substitutions, the structural homogeneity and stability of

such models are indispensable prerequisites. It is also very important that the chosen model

system and its analogues are easy to synthesize. Although successful attempts at incorpo-

rating ﬂ uorinated amino acids by diverse protein expression methods have been reported

[17, 18] , most synthetic strategies for peptides bearing nonnatural substitutions rely on

solid - phase peptide synthesis (SPPS). While linear SPPS is restricted by the achievability

of long sequences [19] , convergent synthetic routes applying various peptide ligation

methods [20] as well as expressed protein ligation [21] pave the way to large modiﬁ ed

proteins. Nevertheless, fast synthetic approaches are desirable for the synthesis of a broad

variety of different modiﬁ ed peptides. In addition, comparably small model systems allow

for a more comprehensive interpretation of experimental data. Consequently, model

systems are often signiﬁ cantly smaller than natural proteins and, thus, have to be very

carefully designed to efﬁ ciently mimic a natural protein environment. In this respect, α -

helical coiled coil peptides have greatly gained in importance in recent years [7] . Coiled

coils are ubiquitous small proteins that show broad biological activities [22] . As the struc-

tural components of many DNA - binding proteins, they play an important role in gene

transcription, cell growth, and proliferation. Larger coiled coil assemblies provide molecu-

lar scaffolds and networks for the cytoskeleton as well as important structural components

of so - called “ motor proteins ” [23] . Such naturally occurring coiled coils are usually com-

posed of two to ﬁ ve monomeric α - helices whose primary structure is characterized by a

repetitive alignment of seven amino acids ( abcdefg )

called a “ heptad repeat ” . Positions

a and d are mostly hydrophobic and harbor leucine, valine, and in some cases isoleucine

and methionine. In the folded state, these positions point to one side of the helix, whereas

the predominantly hydrophilic positions b , c , and f point to the other side. This spatial

separation of hydrophobic and hydrophilic residues imparts signiﬁ cant amphiphilicity to

the molecule. Due to segregation of hydrophobic surface area from the aqueous solvent,

the helices usually fold into left - handed superhelical oligomers that bury the hydrophobic

residues within the so - called “ hydrophobic core ” (Figure 15.1 ). In dimeric coiled coils,

Application of Artiﬁ cial Model Systems 393

positions e and g are usually occupied by charged residues such as glutamic acid, lysine,

or arginine. These residues additionally stabilize the folded structure by attractive interheli-

cal coulomb interactions. Besides its stabilizing role, this charged interaction domain is

an important determinant of folding speciﬁ city [24] . This detailed knowledge about their

structure enables the de novo design of coiled coils that predictably fold into a speciﬁ c

oligomeric structure that is best suited for a certain investigation. In the subsequent

sections, we will summarize how the coiled coil can be used to study ﬂ uorine as a

side - chain substituent in different native - like polypeptide environments. The incorporation

of ﬂ uorinated amino acids at different sites within the heptad repeat allows one to probe

the impact of ﬂ uorination in both hydrophobic and polar environments. Furthermore,

the position of ﬂ uorinated residues as well as the folding speciﬁ city of the coiled coil

determines the orientation of the side - chains. Accordingly, the interactions of ﬂ uorinated

amino acids depend not only on the nature of the environment but also on the way they

“ look ” at it.

Figure 15.1 Schematic representation of a modelled antiparallel coiled coil homodimer in

both a side view (top) and a view along the superhelical axis (bottom). Hydrophobic side -

chains (Leu) are represented in yellow, complementary salt bridges in red (Glu), and blue

(Lys or Arg). See color plate 15.1.

394 Fluorine in Medicinal Chemistry and Chemical Biology

15.2 Hydrophobicity, Spatial Demand and Polarity – Fluorine in a

Hydrophobic and a Polar Polypeptide Environment

A rationally designed antiparallel, homodimeric α - helical coiled coil peptide served as a

model system for the ﬁ rst systematic approach to investigating the interaction character-

istics of ﬂ uorinated amino acids with native residues (Figure 15.2 ). The hydrophobic core

of the model is exclusively composed of leucine, whereas complementary coulomb inter-

actions between positions e and e ′ as well as g and g ′ control the antiparallel alignment

of the helices within the dimer [25] . In both strands, position a9 in the hydrophobic core

Figure 15.2 Helical wheel and sequence representation of the antiparallel, dimeric model

system. The substitution positions are highlighted in one strand with an open square for the

hydrophobic core and an open circle for the charged domain. Their direct interaction partners

in the opposite strand are shaded in gray squares or circles, respectively. The ligation site is

marked with an arrow.

Application of Artiﬁ cial Model Systems 395

and position g8 in the charged interface served as substitution positions for ﬂ uorinated

amino acids. This substitution pattern enabled an investigation of the effects of ﬂ uorination

in both a hydrophobic and a hydrophilic coiled coil environment. The antiparallel orienta-

tion of the two 41 - amino - acid peptide chains ensures that ﬂ uorinated residues interact

exclusively with native residues in the opposite strand. As the peptides were to be used

in an assay of the self - replication rate of these peptides (see next section), a convergent

synthesis strategy based on native chemical ligation [26] was applied. Three f - positions

contain tyrosine as an analytical label.

The strength of hydrophobic interactions largely correlates with the hydrophobic

surface area but also depends on packing effects of the interacting residues. Regarding the

hydrophobic core of the coiled coil model, the ﬁ rst important step was to choose the amino

acids that would be appropriate for a systematic study of the impact of ﬂ uorination. Single

ﬂ uorine substitutions for hydrogen often behave bio - isosterically to hydrogen [27] , that

is, they alter neither the conformation nor the activity of the molecule. Nevertheless, there

is no linear correlation between the degree of ﬂ uorination and the spatial demand of ﬂ uo-

rinated alkyl groups. A comparison of the van der Waals volumes shows a triﬂ uoromethyl

group (42.8 Å

) to be approximately twice as large as a methyl group (24.5 Å

). These

volumes are calculated on a per - molecule basis according to published procedures [28] .

Furthermore, the steric effects of a triﬂ uoromethyl group, which are determined by both

spatial demand and conformational ﬂ exibility, show similarities to an isopropyl group [29]

rather than to a methyl group.

Accordingly, the investigations were based on ( S ) - aminobutyric acid (Abu) deriva-

tives. Stepwise ﬂ uorination of the Abu side - chain yields ( S ) - diﬂ uoroethylglycine (DfeGly)

and ( S ) - triﬂ uoroethylglycine (TfeGly). The spatial demand of the side - chain can be further

increased by elongation by one methyl group, yielding ( S ) - diﬂ uoropropylglycine (DfpGly),

which may be expected to have a spatial demand that is comparable to that of leucine.

Alanine, with the smallest side - chain in this series, served as a control substitution. Figure

15.3 summarizes the discussion of steric size/effects and the resulting substitution

pattern.

The analysis of temperature - induced unfolding monitored by circular dichroism (CD)

spectroscopy yields the midpoint of thermal denaturation. The melting point ( T

) is deﬁ ned

as the temperature at which 50% of the oligomer is unfolded. Given that the coiled coil

maintains its folding speciﬁ city when single substitutions are performed, this melting point

serves as a qualitative, yet not absolute, measure of stability. Small differences in T

do not

necessarily imply a difference in thermodynamic stability. Signiﬁ cant changes, however,

may be interpreted in terms of stabilization or destabilization when comparing structurally

equivalent coiled coils since, in many cases, an increase in T

is associated with an increase

in thermodynamic stability [30] . Figure 15.4 shows the melting curves of all variants that

carry substitutes for leucine at position a9 within the hydrophobic core. The respective

melting points are given in Table 15.1 . With the exception of the DfpGly - variant, the

thermal stability in this series of dimers increases along with the increasing spatial demand

of the side - chain in position a9 in the order Ala > Abu > DfeGly > TfeGly > Leu.

Several factors have to be taken into account for a conclusive interpretation of these

results. Recent investigations have shown that some ﬂ uorinated amino acids exhibit less

favorable helix - forming propensities [31] . Thus, structural perturbations of the helical

backbone may contribute to some extent to the general destabilization upon incorporation

396 Fluorine in Medicinal Chemistry and Chemical Biology

Figure 15.3 Based on the proposed comparability of isopropyl and triﬂ uoromethyl, one

native leucine residue within the hydrophobic core is substituted by amino acids bearing ﬂ uo-

rinated ethyl side - chains. Starting from Abu the spatial demand was increased by stepwise

ﬂ uorination and elongation of the side - chain from the left to the right. Alanine served as a

control substitution. The van der Vaals volumes in parentheses were calculated according to

reference [28] and refer to the group attached to the β - carbon of the side - chain.

Figure 15.4 Melting curves of the coiled - coil dimers substituted in position a9 . Parent

peptide (Leu) (

), Ala ( 䊉 ), Abu ( 䊊 ), DfeGly ( 䉭 ),TfeGly ( ⵧ ), and DfpGly ( 䉫 ) .Recorded at

peptide concentration of 20 µ M at a pH of 7.4 (phosphate buffer containing 5 M GdnHCl).

Application of Artiﬁ cial Model Systems 397

of ﬂ uorinated residues. However, with the exception of TfeGly these effects have not been

studied for the amino acids used here. Moreover, helix - forming propensity is not the sole

determinant of the stability of the coiled coil folding motif whose structure formation is

based on intermolecular interactions. The thermodynamic driving force for the oligomer-

ization of coiled coils largely originates from hydrophobic interactions and side - chain

packing effects within the hydrophobic core. Due to such stabilizing intermolecular inter-

actions, the effects of helical propensity are often smaller within the interface of coiled

coils then they are in single helices [32] . In addition, many investigations have shown that

helix - forming propensity can only partly account for conformational preferences of amino

acids and that other interactions such as hydrophobic side - chain packing may also play a

key role in this regard [33] . For example: the helix propensity for Abu is 1.22 while the

value for TfeGly is only 0.05 [31] . Nevertheless, the thermal stability of the folding motif

presented here increases upon replacing Abu by TfeGly. The increase in surface area and

hydrophobicity upon stepwise ﬂ uorination of the Abu side - chain has a favorable effect on

hydrophobic interactions and thus appears to stabilize the folding motif. The DfpGly

variant is excluded from this trend as, surprisingly, it represents the most destabilizing

substitution. Although DfpGly bears a side - chain closest to leucine regarding steric size,

its presence in the hydrophobic interior results in an even stronger destabilization than that

caused by alanine in the same position.

This effect can be ascribed to the highly polarized methyl group at the γ - carbon

(Figure 15.3 ). Even though the side - chain should be bulky enough to gain sufﬁ cient

packing within the hydrophobic core, its polarity appears to prevent a favorable interaction

in this nonpolar environment. The interpretation of this result gains support from two other

ﬁ ndings. The formal γ - diﬂ uorination of the Abu side - chain, although it increases the spatial

demand, shows only a marginal effect on T

( ∆ T

= +0.4 K). One could argue that the

stabilizing effect of increased steric bulk is offset by the destabilization that is introduced

by a more polarized γ - hydrogen atom. Furthermore, the TfeGly substitution, although

expected to show steric effects comparable to leucine, results in a dimer that is roughly

15 K less stable than the parent peptide. As the strong inductive effect of ﬂ uorine affects

the β - methylene groups, this ﬁ nding can also be explained by the polarization of hydrogen

atoms within the side - chain.

Nevertheless, another important aspect regarding the stability of the folded state must

be taken into consideration. In comparison to leucine, TfeGly may exhibit a comparable

spatial demand but it lacks two carbon atoms within the side - chain. Therefore, the con-

formational ﬂ exibility and, consequently, the entropy and stability of the folded dimer are

Table 15.1 Melting points of the Leu9 variants

Amino acid at position a9 T

( ° C)

Leu 73.9

Ala 53.2

Abu 54.0

DfeGly 54.4

TfeGly 59.9

DfpGly 51.6

398 Fluorine in Medicinal Chemistry and Chemical Biology

reduced. However, as the thermal stability of the model system decreases along with the

increasing number of polarized hydrogen atoms buried within the hydrophobic core, the

interpretation in terms of polarity is conclusive. The stability follows the order TfeGly > -

DfeGly > DfpGly. DfpGly bears the highest number of polarized hydrogen atoms and,

accordingly, represents the most destabilizing substitution.

The next important step was to investigate how the same amino acids would behave

as substitutes for lysine in position g8 of the charged domain. This substitution replaces

the salt - bridge to glutamic acid in the opposite helix by introducing ﬂ uorinated amino

acids as noncharged interaction partners. As shown in Figure 15.5 , the resulting impact

on the thermal stability of the dimer is much lower than that observed for the hydrophobic

core (and see Table 15.2 ).

The decrease in melting temperature compared to the Lys - variant ranges from 2 K

for Abu to roughly 6 K for DfeGly. There is no clear trend pointing to a correlation between

solvent - exposed hydrophobic surface area and thermal stability. However, there are very

interesting effects arising from ﬂ uorination of the Abu side - chain. Fluorine substitutions

in position g8 generally result in a somewhat stronger destabilization than the incorpora-

tion of Ala and Abu. As shown for substitutions within the hydrophobic core, ﬂ uorinated

Figure 15.5 Melting curves of the coiled coil dimers substituted in position g8 . Parent peptide

(Lys) (

), Ala ( 䊉 ), Abu ( 䊊 ), DfeGly ( 䉭 ),TfeGly ( ⵧ ), and DfpGly ( 䉫 ) . Recorded at peptide

concentrations of 20 µ M at pH 7.4 (phosphate buffer in 5 M GdnHCl).

Table 15.2 Melting points of the Lys8 variants

Amino acid at position g8 T

( ° C)

Lys 73.9

Ala 71.3

Abu 71.9

DfeGly 68.3

TfeGly 68.9

DfpGly 68.6

Application of Artiﬁ cial Model Systems 399

alkyl groups exhibit the unique property of being polar and hydrophobic. This “ polar

hydrophobicity ” [34] of ﬂ uorinated compounds is one reason for the preferential formation

of ﬂ uorous phases. Following this argument, the ﬂ uorine - substituted peptides may unfold

more readily than their nonﬂ uorinated analogues to enable ﬂ uorous interactions between

the unfolded peptide chains. This assumption gains support from investigations on the

self - replication properties of the model system [9] presented in the next section.

15.3 Do Fluorine – Fluorine Interactions Interfere with Coiled Coil

Association and Dissociation?

The analysis of temperature - induced unfolding as described in the preceding section

explains how single substitutions of proteinogenic residues by ﬂ uorinated amino acids

affect the dimer ’ s overall stability. The study of the impact of ﬂ uorine substitutions on the

replicase activity of coiled coils provides some valuable additional insight. Primary struc-

tures based on the heptad repeat motif exhibit the ability to promote the condensation of

two monomeric fragments whose amino acid sequence follows a complementary heptad

repeat. Thus, depending on the primary structure of the fragments, coiled coil peptides

can act as either ligases or replicases [35] . The investigations presented in this section are

based on the replicase activity, which explains the convergent route for the synthesis of

the model peptide (see Section 15.2 ).

For the establishment of such a replicase cycle, two peptide fragments are required,

one of which carries a cysteine at its N - terminus (nucleophilic fragment). The electrophilic

fragment usually carries a C - terminal benzyl thioester [36] . A noncatalyzed peptide bond

formation between the nucleophilic and the electrophilic fragment that yields a full - length

monomer initiates the process of self - replication. The ﬁ rst step in this reaction is a thioes-

ter - exchange reaction between the C - terminal benzylthioester moiety of the electrophilic

fragment and the N - terminal cysteine of the nucleophilic fragment followed by an irrevers-

ible S → N acyl migration to yield a native peptide bond (native chemical ligation) [26] .

Based on the complementary coiled coil interactions, the monomer catalyzes the ﬁ rst cata-

lytic cycle, acting as a template for the annealing of the nucleophilic and the electrophilic

fragments. The speciﬁ c association of the fragments with the template brings their reactive

functional groups into close proximity and thereby promotes the fragment condensation

as described above. The cycle is ﬁ nalized by the dissociation of the oligomer, releasing a

newly formed template for further reaction cycles. For the investigation of replicase activ-

ity, the proteinogenic residues in both substitution positions ( g8 and a9 ) of the model

peptide were replaced by three different amino acids that bear a side - chain of identical

length and vary only in ﬂ uorine content (Abu, DfeGly, and TfeGly). Figure 15.6 shows

the turnover for peptides that contain these residues within the hydrophobic as well as

within the charged interaction domain.

The substitution of Abu for leucine in the hydrophobic core position a9 shows that

a signiﬁ cant decrease in spatial demand and hydrophobicity that thermally destabilizes the

folding motif also reduces the rate of product formation. The γ - di - and triﬂ uorination

of Abu further decelerates rather than accelerates the reaction. This trend correlates

with the number of ﬂ uorine atoms within the side - chain. Interestingly, the same kind of