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

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400 Fluorine in Medicinal Chemistry and Chemical Biology

substitutions in the charged domain yields comparable results. In general, the removal of

one salt - bridge of the charged domain slightly accelerates the reaction, as shown for the

substitution of Abu for Lys in position g8 . The association of coiled coils is thermodynami-

cally driven by the formation of the hydrophobic core. Since for substitutions in the

charged interface the hydrophobic core remains unchanged, the association is presumably

not affected. Therefore, the acceleration upon the incorporation of Abu into position g8

may be interpreted as an increased dissociation rate in the last step of the cycle. The sub-

sequent ﬂ uorination, however, appears to decrease the rate of product formation. Again,

this trend correlates with the ﬂ uorine content.

According to the unfolding experiments (see preceding section), the stability of

hydrophobic interactions follows the order Abu < DfeGly < TfeGly. However, in the

charged domain of the dimer the ﬂ uorinated residues generally represent the more desta-

bilizing substitutions compared with the non - ﬂ uorinated residues. Most importantly, the

decrease in replication rate follows the same order as for substitutions in the hydrophobic

core (Abu > DfeGly > TfeGly). Being hydrophobic and lipophilic, the ﬂ uoroalkyl groups

in the unfolded peptides segregate from both the water and hydrophobic coiled coil resi-

dues. This ﬂ uorous effect is comparable to the well - known hydrophobic effect that is

accompanied by a lipophobic effect for partly ﬂ uorinated alkyl groups [10] . A plausible

explanation would be that ﬂ uorinated amino acids exhibit ﬂ uorine – ﬂ uorine ‘ interactions ’

in the unfolded state of the peptides and therefore interfere with folding. A similar expla-

nation has also been proposed as the reason for the destabilization of chloramphenicol

acetyltransferase upon global ﬂ uorination [37] . Fluorous ‘ interactions ’ may indeed be

rather speciﬁ c. The association of transmembrane helices in lipid bilayers, for example,

is much more efﬁ cient when native leucine residues are replaced by hydrophobic and

lipophobic ﬂ uorinated analogues [38] . Accordingly, the same effect may be responsible

for inhibiting the coiled coil association in the ﬁ rst step of the replicase cycle. It may be

argued that, as a contrary effect, the dissociation within the last step of the cycle should

consequently be enhanced. Nevertheless, the ﬂ uorous effect of a single amino acid is very

likely to be more efﬁ cient within the short fragments so that the effect on the association

rate is much more pronounced. Figure 15.7 provides an image of how, following the argu-

Figure 15.6 Time dependent turnover of the fragments substituted in position a9 (left panel)

and g8 (right panel). The turnover vs. time plot is shown for the parent peptide (

) and its

Abu ( 䊊 ), DfeGly ( 䉭 ), and TfeGly ( ⵧ ) substituted analogues.

Application of Artiﬁ cial Model Systems 401

ments above, we assume the ﬂ uorous effect to compete with coiled coil formation and,

thus, to alter replicase activity.

The ﬁ ndings gained with the model system show that two contrary effects character-

ize the interactions of ﬂ uorinated amino acids within the hydrophobic core: spatial demand

and hydrophobicity on one side, and ﬂ uorine - induced polarity on the other. While the

increase in hydrophobic surface area upon ﬂ uorination may be favorable for hydrophobic

interactions, ﬂ uorine ’ s inductive effect appears to interfere with the formation of an intact

hydrophobic core. In addition, the investigations of ﬂ uoroalkyl side - chains in the charged

domain as well as the analysis of ﬂ uorine ’ s effect on replicase activity indicate that

contacts between ﬂ uorinated residues may also have an impact on peptide and protein

folding.

The antiparallel homodimeric model system described above, however, represents a

rather speciﬁ c example. Although the ﬂ uorinated amino acids here interact exclusively

with native residues, two ﬂ uorinated residues are present in the folded state due to homodi-

mer formation. The next goal was to study how subtle changes in the environment of the

ﬂ uorine substitution – that is, its position within the hydrophobic core – affect its impact

on folding and stability. The investigations described in the next section represent a further

important step.

Figure 15.7 The rate of replication depends on the association – dissociation equilibrium. The

data for time - dependent turnover raise the question whether ﬂ uorous interactions interfere

with the association of the electrophilic fragment with the template thereby inhibiting catalysis

[9] . See color plate 15.7.

402 Fluorine in Medicinal Chemistry and Chemical Biology

15.4 Effects of Fluorination Are Difﬁ cult to Predict Because the Impact

of Spatial Demand, Hydrophobicity, and Polarity on Structural Stability

Depends on the Environment

A new coiled coil model system was designed so that the ﬂ uorinated peptide interacted

with an exclusively native interaction partner that, itself, was not intrinsically affected by

ﬂ uorine. Therefore, the folding speciﬁ city of the coiled coil system had to be reversed by

design. The question was whether the ﬁ ndings for this new model system would be com-

parable to those for the antiparallel model system described in the preceding sections.

As mentioned before, coiled coil folding speciﬁ city can be directed by design. Peptide

chains based on the heptad repeat can fold into homo - or heterooligomers either in a paral-

lel or antiparallel fashion. For the new model system, homooligomerization had to be

prevented to make ﬂ uorine – ﬂ uorine contacts impossible. This condition was achieved by

designing two complementary peptides [39] . Peptide VPE presents negative charges (glu-

tamic acid) at its e - and g - positions while peptide VPK presents positive charges (lysine)

at the same positions. Under physiological conditions (pH 7.4), heterodimerization is

favored because the Coulomb interactions between these positions are repellant in a

homodimeric arrangement of VPE and VPK, respectively. Since in this case the charged

positions were needed to control heterodimerization, they could not be used to control the

orientation of the dimer as well. Thus, a redesign of the hydrophobic core was necessary.

The comparison of naturally occurring heptad repeats shows that β - branched amino acids

such as valine are highly conserved in a - positions of parallel coiled coil dimers but that

they are barely found in their antiparallel dimeric counterparts [40] . Placing valine at a -

and leucine at d - positions, therefore, favors a speciﬁ c, parallel orientation of the dimer.

Peptide VPE was used as a template to screen various ﬂ uorinated variants of VPK. In this

study, the ﬂ uorinated amino acids and Abu (see Figure 15.3 ) were incorporated into the

hydrophobic core either at position a16 or at position d19 of VPK (Figure 15.8 ). Two dif-

ferent hydrophobic core positions were chosen to study the impact of the immediate

environment of the substitution on the interactions of the ﬂ uorinated side - chains. Two

peptides that contain leucine instead of ﬂ uorinated amino acids at the respective substitu-

tion position served as the reference peptides.

The stability of all dimers was assessed by temperature - induced unfolding experi-

ments monitored by circular dichroism spectroscopy in the absence of guanidinium hydro-

chloride. It was therefore possible to derive the standard free energy of unfolding from

data ﬁ tting using the Gibbs – Helmholtz equation (15.1) adapted to a two state monomer –

dimer equilibrium.

∆∆ ∆GH

cT T T

RT K

°= −













+−−

























−

m m

1lnln

(15.1)

K is deﬁ ned in terms of fraction unfolded f

by equation 15.2 :

[]

−

(15.2)

Application of Artiﬁ cial Model Systems 403

with [D]

being the concentration of the dimer at the starting temperature where the

peptides were assumed to be fully folded. The change in heat capacity ∆ C

was calculated

to be 0.94 ± 0.1 kcal/mol [39] .

Figure 15.9 shows the ﬁ tted melting curves as a plot of fraction unfolded against

temperature for the peptides substituted at position a16 . The respective melting points and

standard free energies of unfolding are given in Table 15.3 .

While the di - and triﬂ uorination of the Abu - side - chain have only marginal effects on

the stability of folding, the bulky DfpGly side - chain stabilizes the structure by roughly

0.8 kcal/mol. Thus, a signiﬁ cant increase in spatial demand of the ﬂ uorinated side - chain

is able to stabilize the interactions at position a16 . Nevertheless, all peptides are less stable

than the control peptide carrying leucine at this position. Except for DfpGly, these results

are qualitatively in good agreement with those gained for the antiparallel model system.

The signiﬁ cant difference is that DfpGly represents the most stabilizing substitution at

position a16 within this series of ﬂ uorinated peptides. The packing of hydrophobic side -

chains has a much stronger impact on the stability of antiparallel coiled coils than it has

for their parallel analogues because they are more tightly buried within the hydrophobic

Figure 15.8 Helical wheel and sequence representation of the parallel, dimeric model

system. The substitution positions are highlighted in gray. Their main interaction partners are

encircled in black.

404 Fluorine in Medicinal Chemistry and Chemical Biology

Figure 15.9 Melting curves of the coiled coil dimers substituted in position a16 . Leu ( ),

Abu ( 䊊 ), DfeGly ( 䉭 ),TfeGly ( ⵧ ), and DfpGly ( 䉫 ) . Recorded at overall peptide concentra-

tions of 20 µ M at pH 7.4 (phosphate buffer).

Table 15.3 Standard

free energy of unfolding and melting points of the a16

substituted dimers

Amino acid at position a1 6 T

( ° C)

∆ G ° [kcal/mol)

Leu 77.9 13.83

Abu 65.9 11.48

DfeGly 66.0 11.46

TfeGly 69.0 11.51

DfpGly 69.3 12.27

Standard state = 1 M, 101 325 Pa, 25 ° C.

core of antiparallel coiled coils [41] . Obviously, the DfpGly side - chain in position a16 of

the parallel model is more ﬂ exible than it is in position a9 of its antiparallel counterpart.

Consequently, the polarized γ - methyl group of DfpGly is less tightly buried within the

core, which prevents it from disturbing the hydrophobic interactions. The side - chains of

DfeGly and TfeGly are shorter and, thus, less ﬂ exible. Thus, unlike for the γ - methyl group

of DfpGly, the proximity of the polarized β - hydrogen atoms to the rigid backbone does

not allow for a more favorable positioning toward the hydrophobic interaction partners

and, therefore, disturb the formation of an intact hydrophobic core.

The results for substitutions in position d19 additionally support the ﬁ nding that

the orientation of ﬂ uorinated side - chains and the way they are packed against their

interaction partners determines their impact on hydrophobic polypeptide environments.

The substitution of Leu at position d19 by Abu and its ﬂ uorinated analogues shows

effects that are comparable to those in position a16 with respect to loss in unfolding

free energy (Figure 15.10 and Table 15.4 ). The formal diﬂ uorination of Abu slightly

increases the spatial demand and, accordingly, the stability of the coiled coil dimer.

Application of Artiﬁ cial Model Systems 405

Figure 15.10 Melting curves of the coiled coil dimers substituted in position d19 . Leu ( ),

Abu ( 䊊 ), DfeGly ( 䉭 ),TfeGly ( ⵧ ), and DfpGly ( 䉫 ) . Recorded at overall peptide concentra-

tions of 20 µ M at a pH of 7.4 (phosphate buffer).

Table 15.4 Standard

free energy of unfolding and melting points of the d19

substituted dimers

Amino acid at position d19 T

( ° C)

∆ G ° (kcal/mol)

Leu 71.3 11.66

Abu 53.7 9.64

DfeGly 56.9 9.99

TfeGly 55.3 9.87

DfpGly 57.5 10.00

Standard state = 1 M, 101 325 Pa, 25 ° C.

The incorporation of an additional ﬂ uorine substituent (TfeGly) or a methyl group (DfpGly),

however, shows only marginal effects. The ﬁ nding for DfpGly, again, is in sharp

contrast not only with the ﬁ nding for the antiparallel coiled coil but also with results

for position a16 within the same peptide. At position a16 the additional methyl group

was able to stabilize the structure, while this effect is much less pronounced at position

d19 .

Unfortunately, no high - resolution structures of the ﬂ uorine containing coiled coil

dimers are available at present. Nevertheless, a feasible explanation for the different results

for the two substitution positions a16 and d19 can still be found by analyzing the high -

resolution coiled coil structure of a sequence - equivalent interaction motif, the GCN4

leucine zipper [42] . A comparison of the a - and d - positions within the crystal structure of

GCN4 reveals important differences in terms of side - chain packing. The side - chains in

position a pack parallel against the side - chains in position a ′ , while those in position d pack

against position d ′ of the opposite strand. The differences between positions a and d arise

from the C

– C

vectors of the side - chains pointing in different directions. In the case of

406 Fluorine in Medicinal Chemistry and Chemical Biology

position a , this vector points away from the hydrophobic core as well as from the interaction

partner in the opposite strand. In contrast, the vectors point into the core and toward their

interaction partners within the opposite strand for d - positions (Figure 15.11 ).

This difference in side - chain orientation highly affects the packing as well as the

interactions of the ﬂ uorinated side - chains with their proteinogenic counterparts. The amino

acids studied here all share one similarity: they all carry ﬂ uorine atoms at the γ - carbon

atom. Consequently, they strongly polarize the β - methylene groups that point in different

directions – away from the interaction partner for the a - position and toward it for the d -

position. It is plausible that, accordingly, ﬂ uorine - induced polarity at the β - methylene

groups has a stronger impact at the d - position of the parallel model system. These argu-

ments may explain why a signiﬁ cant increase in spatial demand from TfeGly to DfpGly

has the potential to stabilize the interactions within the a - position while it seems impossi-

ble at the d - position.

These ﬁ ndings indicate that although ﬂ uorine may impart unique and general proper-

ties to hydrocarbons, these properties can have different effects at different positions within

a hydrophobic protein environment. Coiled coils share a unique and very speciﬁ c interac-

tion pattern. Nevertheless, their stability is susceptible to subtle structural modiﬁ cations,

which makes them valuable candidates for systematically investigating the effects of ﬂ uo-

rine on peptide – protein interactions.

Figure 15.11 Schematic representation of the side - chains at a - and d - positions within the

hydrophobic core of the parallel GCN4 coiled coil dimer (according to reference [42] ). The

asterisk marks the β - carbon atoms that would be polarized in the case of the ﬂ uorinated amino

acids used for these investigations.

Application of Artiﬁ cial Model Systems 407

15.5 Summary and Prospects

The studies reviewed in this chapter describe the impact of single ﬂ uorinated amino acid

substitutions on protein stability in terms of hydrophobic as well as polar interactions

within coiled coil - based model systems. Aiming at a general description of possible

molecular interactions of ﬂ uorinated amino acids within native protein environments, we

can summarize our results as follows.

In contrast to their canonical counterparts, ﬂ uorinated amino acids uniquely combine

two contrary properties within their side - chain: hydrophobicity and polarity. Thus, ﬂ uori-

nated analogues of hydrophobic amino acids affect peptide and protein stability in a way

that is not easily predictable, because it is difﬁ cult to evaluate the impact of each effect

separately. Nevertheless, in contrast to global ﬂ uorination of protein interaction domains,

single amino acid substitutions generally have a destabilizing effect on the hydrophobic

as well as the charged interface of coiled coil peptides. Most importantly, the environment

of the substitution itself plays a key role in this context. In these studies, the environment

is deﬁ ned by either a parallel or an antiparallel orientation of coiled - coil dimers as well

as by the substitution position, either in the hydrophobic core or in the charged interaction

domain. The speciﬁ city of coiled coil folding determines the orientation of amino acid

side - chains, which results in divergent impacts of the dipoles that are induced by ﬂ uorine

substituents ( a - versus d - position). Accordingly, a general prediction of the inﬂ uence of

ﬂ uorinated amino acids on the nature and stability of protein folding seems to be difﬁ cult

at present.

The model systems described here deliver useful information about the effect of

single ﬂ uorinated amino acids on coiled coil folding but, due to the versatility of natural

folding motifs, individual proof of structural and thermodynamic effects caused by incor-

poration of ﬂ uorinated amino acids is needed in order to utilize their full potential.

A future goal would be to screen for ﬂ uorophilic interaction partners within the pool

of the proteinogenic amino acids to guide an optimal application of ﬂ uorinated analogues

in peptide and protein engineering.

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