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

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Fluorinated Amino Acids and Biomolecules in Protein Design and Chemical Biology 421

for therapeutics. However, it is challenging to obtain structural information on these pro-

teins, as they are difﬁ cult to crystallize or analyze by solution NMR methods.

F NMR

studies in lipid bilayers have advanced our understanding of both membrane proteins and

antimicrobial peptides. In one approach, residues in the TM1 domain (residues 32 – 48) of

diacylglycerol kinase (DAGK) were mutated to Cys, which were then thioalkylated with

3 - bromo - 1,1,1 - triﬂ uoropropanone [61] . Oxygen is distributed nonhomogeneously in the

membrane, and exerts an inﬂ uence on the chemical shift of ﬂ uorine, hence allowing for

determination of the depth of the residue within the bilayer. The DAGK constructs were

assembled in micelles and the 1D solution - state

F NMR spectrum was obtained in the

presence of ambient and increased O

partial pressures. The change in chemical shift

induced by oxygen ( ∆ σ

) was plotted for each residue. The resultant plot showed an oscil-

lation of ∆ σ

with a period of 3.6 residues, suggesting that this domain forms an α - helix.

Further, the magnitude of changes of oxygen - induced chemical shifts indicated that only

one side of the helix interfaces with the lipids.

Antimicrobial peptides such as magainin form cationic and hydrophobic domains

upon α - helix formation. These peptides are attracted to anionic cell surfaces and insert

their hydrophobic domains into the lipid bilayer, eventually compromising the integrity of

the membrane [62] . There are several proposed mechanisms of action for this class of

antimicrobial peptides. In the “ carpet mechanism, ” the α - helical axis is parallel to the

membrane surface (S - state) and the peptide is monomeric. In the “ barrel - stave ” and “ toroi-

dal pore ” mechanisms, the axes of α - helices axis are perpendicular to the membrane

surface (I - state). Ulrich and co - workers have used

F NMR to determine the orientation

of helices in the membrane [63] . The strength of

F dipolar coupling is large, and distances

upto 17.5 Å can be measured assuming a resolvable 30 Hz homonuclear coupling [64] . A

series of peptide variants of PGLa, peptidyl - glycylleucine - carboxylamide, a member of

the magainin family with 4 - CF

- phenylglycine substitutions were synthesized. Residual

dipolar coupling between

F nuclei was used to determine helix orientation in a macro-

scopically aligned lipid environment. At low concentrations (peptide/lipid molar ratio

∼ 1 : 200), PGLa sits parallel to the membrane surface (90 ° , S - state). However, at peptide

concentrations (peptide/lipid molar ratio > 1 : 50) required for activity, PGLa dimerizes

in a tilted orientation (120 ° , T - state) (Figure 16.4 ). This newly discovered T - state may

represent an intermediate between the S - state and I - state and is also supported by

NMR.

Hong and co - workers have used

F NMR to investigate the antimicrobial peptide

protegrin - 1 (PG - 1) in lipid bilayers. They ﬁ rst determined that PG - 1 forms a dimer in 1 -

palmitoyl - 2 - oleoyl - sn - glycero - 3 - phosphocholine (POPC) bilayers using a

F CODEX

(centerband - only detection of exchange) method, evident by two 4 - F - Phe12 residues that

were within a 15 Å distance [65] . However, the dimer interface could not be described in

sufﬁ cient detail. Modeling of the PG - 1 dimer was based on its solution structure (PDB

code: 1PG1) and indicated that the F– F distance in both the parallel and antiparallel dimers

is within 11 – 14 Å . Hong then employed resonance - echo double - resonance (REDOR)

solid - state NMR [66] in an attempt to resolve this orientational ambiguity. Phe12 in PG - 1

was labeled with

F, Val16 with

C, and Cys15 with

N and

C. Several intermolecular

and intramolecular

F –

C, or

H –

N –

C distances were obtained from both experi-

mental REDOR data ﬁ tting and modeling of PG - 1. It was determined that PG - 1 adopts

a parallel dimer in POPC (Figure 16.5 ), where the C - terminal regions form the dimer

422 Fluorine in Medicinal Chemistry and Chemical Biology

Figure 16.4 Illustration of the S - state and T - state of the antimicrobial peptide PGLa in a

DMPC membrane. At low concentrations, PGLa adopts an S - state. At high concentrations,

PGLa assumes a tilted T - state. PGLa forms a dimer in the T - state (shown in purple). See color

plate 16.4.

( Source: Glaser, R. W., Sachse, C., Durr, U. H. N. et al. , Concentration - dependent realignment

of the antimicrobial peptide PGLa in lipid membranes observed by solid - state F - 19 - NMR,

Biophys. J. (2005) 88 , 3392 – 3397. Reproduced with permission from the Biophysical

Society.)

interface. The design of membrane - disrupting antimicrobial peptides could beneﬁ t from

the identiﬁ cation and characterization of the dimer interfacial region.

16.3.4

F Amino Acids as PET Tracers

Reliable methods for early identiﬁ cation of tumors are crucial for successful treatment

regimens. Positron emission tomography (PET) is used to detect radiolabeled compounds

that have speciﬁ city for tumors [67, 68] . The half - life of

F ( ∼ 110 min) is longer than

those of other PET isotopes such as

N, and

O. This makes it a popular choice, since

radioactive samples may be synthesized and tranferred to a PET facility, allowing greater

access to this technology. By far the most common tracer in oncology is 2 - [

F]ﬂ uoro - 2 -

deoxy - d - glucose; however,

F - labeled amino acids have also shown promising results in

recent studies [69] . The uptake of amino acids by cancer cells is faster than that by normal

cells [70, 71] , as active transport systems are upregulated in tumor cells [71] . Studies with

( O - (2 - [

F]ﬂ uoroethyl - l - tyrosine) showed higher speciﬁ city for uptake by tumors than for

other conditions such as inﬂ ammation [72, 73] . The amino acid l - DOPA is important for

brain function and [

F]ﬂ uoro - l - DOPA has been used to study dopamine synthesis – an

important factor in diseases such as Parkinson ’ s [74] .

16.3.5 Fluorinated Aromatic Amino Acids in Receptors and Enzymes

Fluorinated aromatic amino acids have been used to identify and characterize cation – π

molecular recognition events in biology. This type of interaction often occurs in protein –

cation ligand binding pairs. Addition of ﬂ uorine(s) to aromatic rings decreases the negative

electrostatic potential on the ring and weakens cation – π interactions. Systematic mutation

of aromatic (Phe, Tyr, Trp) residues in proteins with ﬂ uorinated analogues and subsequent

Fluorinated Amino Acids and Biomolecules in Protein Design and Chemical Biology 423

Figure 16.5 The dimer structure of PG - 1 (PDB code: 1ZY6) in POPC bilayers as determined

by solid - state NMR. Isotopically labeled amino acids are shown in stick format; Phe12 was

labeled with

F, Cys12 with

N and

C, and Val16 with

C. Inter - and intramolecular

distances were used to determine the relative position of the two monomers. See color

plate 16.5.

424 Fluorine in Medicinal Chemistry and Chemical Biology

functional assays have provided insights in identifying cation – π interactions and mapping

the binding sites in ligand – receptor pairs that utilize such interactions [75] .

The nicotinic acetylcholine receptor (nAChR) belongs to a superfamily of ligand -

gated ion channels that includes glycine, 5 - hydroxytryptamine - 3A (5HT

), and γ - amino-

butyric acid (GABA) receptors. These proteins are pentameric, with ﬁ ve homologous

subunits arranged around a central pore. Ligand binding sites are located on the extracel-

lular N - terminal domains. Of particular interest are a number of aromatic residues near

the ligand - binding site. To assess whether these aromatic side - chains are involved in the

recognition of the quaternary ammonium group of acetylcholine (ACh), Dougherty and

co - workers incorporated a series of ﬂ uorinated tryptophan derivatives into four Trp sites

( α 86, α 149, α 184, and γ 55/ δ 57) in nAChR [76] . These ﬂ uorinated aromatic residues are

introduced into receptors through the site - directed nonsense suppression method [51] . The

successful heterologous expression of mutated receptors onto the cell surface allowed for

subsequent electrophysiological investigations. Tetra - ﬂ uorinated Trp at α 86, α 184, and

γ 55/ δ 57 gave receptor activation (EC

) values that did not differ signiﬁ cantly from wild

type (wt) ( < 2 - fold). This observation indicated that the steric perturbation on the receptor

due to ﬂ uorine substitution is tolerated, and that these sites do not direct strong cation – π

interactions. However, tetra - ﬂ uorinated Trp at α 149 dramatically shifted the EC

value

from 50 µ M (wt) to 2700 µ M. More importantly, the EC

values were strongly correlated

with the level of ﬂ uorination on the indole ring at position α 149. A linear relationship

between log[EC

/EC

(wt)] and the calculated gas - phase cation – π binding afﬁ nity was

established (Figure 16.6 ) [76] . These results suggest that the ammonium group of ACh

makes van der Waals contact with the indole ring of Trp149. In fact, a constitutively active

receptor resulting from incorporation of

Tyr CH N CH−−

()

−

()

at α 149 validated this

ﬁ nding. Similar experiments have led to Trp183 of the 5 - hydroxytryptamine - 3A receptor

(5HT

R) being implicated in a cation – π interaction with the primary ammonium ion of

serotonin (Figure 16.6 ) [77] . Along these lines, ﬂ uorinated phenylalanine analogues have

been introduced into the GABA

receptor. Functional measurements revealed a novel

cation – π interaction between GABA and Tyr97 in the β

subunit of the receptor [78] .

Stubbe, Nocera, and co - workers have employed ﬂ uorinated tyrosines to probe the

mechanism of proton - coupled electron transfer (PCET) in ribonucleotide reductases

(RNRs) [79] . The E. coli RNR, composed of two subunits (R1 and R2), catalyzes the

conversion of nucleotides to deoxynucleotides. Substrate reduction requires a Cys439

radical in R1, propagated from a Tyr122 radical in R2. Three residues from each subunit

participate in this long - distance ( > 35 Å ) radical propagation [Tyr122 → Trp48 → Tyr356

within R2, then → Try731 → Tyr730 → Cys439 within R1]. Residue Tyr356 is invisible

in the crystal structure of R2 [80] and the docking model of R1 and R2 [81] , but is postu-

lated to be on the radical propagation pathway. To examine its role, Tyr356 in R2 was

replaced by di - , tri - , and tetra - ﬂ uorinated Tyr analogues by the intein - mediated peptide

ligation. The ﬂ uorinated variants retained their reductive activity. In addition, no signiﬁ -

cant difference in reduction rates was observed at a pH at which the variants (p K

∼ 5.6 – 7.8

for Ac - F n Tyr - NH

) are deprotonated but the wild type is not (p K

9.9 for Ac - Tyr - NH

Furthermore, the reduced enzymatic activity was related to the elevated reduction potential

of ﬂ uorinated tyrosines ( E

(Y/Y

−

) ∼ 755 – 968 mV for analogues vs. 642 mV for Ac - Tyr -

) [82] . These measurements point to a redox - active role for Tyr356 and further suggest

that the phenolic proton is not essential in the radical propagation pathway [79] .

Fluorinated Amino Acids and Biomolecules in Protein Design and Chemical Biology 425

16.3.6 Fluorinated Aromatic Amino Acids in Protein Design

The inductive effect of ﬂ uorine on aromatic amino acids can effect not only π – cation

interactions, but also π – π interactions. In addition to enhanced hydrophobicity, perﬂ uori-

nated aromatics exhibit other interesting properties. The solubility parameter values ( δ )

for benzene and perﬂ uorobenzene are similar, 9.2 and

12 32

. cal cm

−

respectively [11] .

This suggests that unlike in the case of hexane and perﬂ uorohexane, these two liquids

should be miscible at room temperature. The ∆ H of mixing is − 1.98 kJ/mol at 25 ° C and

the melting point for crystals of the mixture is about 15 ° C higher than for either pure

component [1] . The powder diffraction map for the mixture indicates that benzene and

perﬂ uorobenzene stack with the H and F atoms aligned. This alignment is quite different

from that proposed in hydrocarbon π – π interactions, where a hydrogen interacts with the

negative quadrupole of the benzene ring. In order to achieve this interaction, the rings can

be aligned perpendicular to one another (an edge – face geometry), or parallel but staggered

(face - to - face). The quadrupole moment for perﬂ uorobenzene (32 × 10

− 40

C m

) is nearly

equal in magnitude and opposite to that of benzene ( − 29 × 10

− 40

C m

). It has been

Figure 16.6 Modulation of cation – π interactions using ﬂ uorinated amino acids. (a) Structures

of acetylcholine (ACh) and 5 - hydroxytryptamine (5 - HT). (b) Receptor activation (log[EC

(wt)]) vs. calculated gas phase cation – π binding ability. Data from references [76] and

[77] .

426 Fluorine in Medicinal Chemistry and Chemical Biology

suggested that a quadrupole interaction is responsible for the observed ∆ H of mixing,

though others have argued that it is a result of coulombic interactions. Gas - phase calcula-

tions suggest that the stacking energy between benzene and hexaﬂ uorobenzene is − 3.7 kcal/

mol at a stacking distance of 3.6 Å [37] . Regardless of the origin of the interaction, its

magnitude suggests that it can be harnessed to stabilize protein folding.

Pentaﬂ uorophenylalanine (f

- Phe) has been incorporated into several constructs, but

clear evidence for π – π stacking has not yet been obtained. Waters and co - workers studied

the effect of f

- Phe on the α - helical stability of a peptide [83] . They designed a series of

peptides with aromatic groups at the i and ( i + 4) positions, so that upon α - helix formation,

the side - chains could interact. Phe – Phe constructs and Phe – f

- Phe constructs showed

similar interaction energies when the aromatic residues were introduced in the center of

the sequence ( ∆ G = − 0.27 kcal/mol). When the aromatic residues were introduced near the

C - terminus, Phe – Phe constructs displayed higher interaction energies ( ∆ G = − 0.8 kcal/

mol) than Phe – f

- Phe constructs ( ∆ G = − 0.55 kcal/mol). Nevertheless, peptides containing

either a Phe – Phe or Phe – f

- Phe interacting pair displayed higher helicities than their

respective control peptides that placed aromatic amino acids at noninteracting positions [ i

and ( i + 5)]. Waters has argued that the Phe – f

- Phe pair cannot achieve ideal geometry for

interaction in this construct, thereby reducing the overall increase in stability of the folded

form.

Gellman and co - workers investigated a 35 - residue peptide, the chicken villin head-

piece subdomain (cVHP), that contains three buried Phe residues. They made mutants in

which one, two, or all of the Phe residues are changed to f

- Phe [84] . They also made

other protein modiﬁ cations to facilitate measurement of the folding free energy. One

mutant, Phe 10 → f

- Phe was more stable than the unmodiﬁ ed sequence by 0.6 kcal/mol.

The NMR solution structure of the Phe 10 → f

– Phe mutant revealed little perturbation in

the geometry of these residues [85] . Fluorinated phenyl rings can be used to stabilize

protein folds, though the stabilizing magnitude might be different from the level expected

from ideal quadrupolar interactions.

It is important to note that the electron - withdrawing effects of ﬂ uorine can inﬂ uence

not only side - chains but also the backbone. Side - chain electron - withdrawing substituents

can perturb the hydrogen - bonding potential of the peptide bond, and alter backbone con-

formation. Using a peptoid construct, Blackwell and co - workers discovered that the place-

ment of ( S ) - N - (1 - (pentaﬂ uorophenyl)ethyl)glycine reduces the stability of a threaded loop

structure that depends upon speciﬁ c hydrogen - bonding interactions [86] , and that the

placement of this residue at the N - terminus results in increased stability. They reasoned

that, by withdrawing electrons, ﬂ uorine increases the acidity of the ammonium ion, hence

increasing its hydrogen - bonding potential. Organic ﬂ uorine is unique in its ability to

perform this task, since other electron - withdrawing atoms (oxygen and nitrogen) could

potentially compete with the hydrogen - bond acceptors and alter structure.

16.3.7 Collagen Stability Revealed Using Fluorine Substitution

Raines and co - workers have used ﬂ uorinated proline to explore stereoelectronic inﬂ uences

on the stability of collagen. Collagen is the most abundant protein in mammals and consists

of three polypeptide chains that form an extended triple helix. Each polypeptide is com-

Fluorinated Amino Acids and Biomolecules in Protein Design and Chemical Biology 427

posed of ∼ 300 (Xaa - Yaa - Gly) repetitive motifs, where Xaa is often proline (Pro) and Yaa

is often 4( R ) - hydroxy - l - proline (Hyp). It has long been known that the 4 - hydroxy group

in Hyp is critical to the stability of collagen and about 10% of residues are Hyp in common

collagen proteins. A crystal structure of collagen [87] revealed that these residues were

involved in interchain hydrogen bonding through structured water molecules. Initially it

was thought that these hydrogen bonds imparted stability to collagen [88] . Raines chal-

lenged this notion, arguing that the entropic cost of sequestering water molecules would

likely be prohibitive in noncrystalline conditions [89, 90] , and further proposed that the

hydroxyl groups might impart stability by imposing conformational constraints on

proline.

To demonstrate that water - mediated hydrogen bonds are not responsible for the

structural stability of collagen, Raines and co - workers used 4( R) - ﬂ uoro - l - proline (Flp) at

Yaa positions to construct collagen mimics. Carbon - bound ﬂ uorine does not form hydro-

gen bonds and is very electronegative – and therefore a suitable probe for investigating

the proposed stereoelectronic effects [90] . Early studies revealed that the C γ - exo ring

pucker is predominant in Hyp residues at Yaa positions. By placing ﬂ uorine at the pro - R

position, a C γ - exo ring conformation should be favored through the “ gauche effect. ” The

C γ - exo ring pucker predetermines the main - chain torsion angles ( ϕ , ψ , ω ) of Flp residues

and they are close to the angles found in collagen (Figure 16.7 ). For example, the ψ angle

in crystalline AcFlpOMe is 141 ° , very close to the main - chain ψ angle of ∼ 150 ° in colla-

gen. Moreover, in such a preorganized ring, an n → π * interaction between O

and C

= O

along the peptide chain occurs [38, 91, 92] . Again, in crystalline AcFlpOMe, the angle

and distance of O

…

= O

are 98 ° and 2.76 Å , reminiscent of the B ü rgi – Dunitz trajec-

tory [93, 94] of nucleophile attack on carbonyl groups (109 ° ± 10 ° and in the range

∼ 1.5 – 3.0 Å ) [95] . This n → π * interaction not only stabilizes the ideal ψ angle for Flp in

the triple helix but also contributes to the required trans amide bond ( ω = 180 ° ) conﬁ gura-

tion. Based on the ability to preorganize the proline ring conformation in order of electro-

negativity F > OH > H, the trend of thermal stability of collagen and collagen mimics

could be predicted. Indeed, Flp stabilized collagen triple helices when incorporated in the

Yaa position, where the T

of (Pro - Flp - Gly)

is 22 ° C higher than that of (Pro - Hyp - Gly)

( T

= 69 ° C) [90] in 50 mM acetic acid ( ∆ T

= 20 ° C in PBS). The T

of (Pro - Pro - Gly)

is 41 ° C. Additional work by Raines ’ laboratory has demonstrated that both 4( S ) - ﬂ uoro - l -

proline (ﬂ p) and 4( S ) - hydroxy - l - Proline (Hyp) at Yaa positions destabilized collagen [38] ,

essentially by imposing a C γ - endo ring pucker. These results demonstrate that stereoelec-

tronic effects are crucial for the extra stability of collagen, and not the bridging water

molecules.

Molecular models indicate that residues at the Xaa position that promote the C γ - endo

conformation would stabilize collagen because of better packing in the triple helical form

[96, 97] . However, substituting Pro at Xaa positions with either C γ - exo constraining Hyp

or C γ - endo constraining Hyp in the (XaaProGly)

construct diminishes their stabilities [96,

97] . One plausible reason could be that hydroxyl groups experience nonbonded steric

interactions in the collagen triple helix. Raines and co - workers again utilized ﬂ uorine to

probe whether there was a stereoelectronic effect in operation at the Xaa position. The

rationale was that − F is smaller than − OH and therefore can be used to avert a potential

steric clash. Residue ﬂ p favors the C γ - endo ring conformation, whereas Flp favors the

C γ - exo ring conformation. As expected, ﬂ p, not Flp, at Xaa positions was able to stabilize

428 Fluorine in Medicinal Chemistry and Chemical Biology

Figure 16.7 Fluorinated prolines in collagen. (a) The trans/cis isomerization of amide bonds

and main - chain angles of proline residues. The n → π * interaction, depicted by a dashed line,

helps stabilize backbone dihedral angles. (b) Electron - withdrawing groups at C γ inﬂ uence the

ring conformation of proline residues through the “ gauche effect. ” The C γ - endo pucker is

favored when R

= H and R

= F, OH, or H. The C γ - exo pucker is favored when R

= OH or

F, and R

= H. Preorganization of the pyrrolidine ring contributes to the thermal stability of

collagen and mimics. Flp: R

= H and R

= F; ﬂ p: R

= F and R

= H; Hyp: R

= H and

= OH; hyp: R

= OH and R

= H. (c) Model structure of collagen (PDB code: 1CAG).

Three hydroxyprolines at Yaa positions from each peptide chain are shown in ball - and - stick

representation. Carbon = gray; Nitrogen = blue; Oxygen = red. See color plate 16.7.

Fluorinated Amino Acids and Biomolecules in Protein Design and Chemical Biology 429

the triple helix. The peptide (ﬂ p - Pro - Gly)

( T

= 33 ° C) forms a triple helix and is more

stable than (Pro - Pro - Gly)

( T

= ∼ 6 – 7 ° C) [96] . The peptide (Flp - Pro - Gly)

does not self -

associate into triple helices. These ﬁ ndings suggest that stereoelectronic perturbations at

the Xaa position can be used to stabilize artiﬁ cial collagen.

Next, both ﬂ p and Flp were introduced into a triplet repeat sequence. The peptide

(ﬂ p - Flp - Gly)

does not form a triple helix [98] . Models suggest that the two ﬂ uorine atoms

abut one another in the folded structure. These clashes can be averted if two strands of

(ﬂ p - Flp - Gly)

are mixed with one strand of (Pro - Pro - Gly)

. With this information in hand,

one can begin to design complex structures that oligomerize in 2 : 1 or 1 : 1 : 1 assemblies.

Natural collagen is diverse in structure. For example, basement - membrane collagen is rich

in 3( R ) - hydroxy - l - proline, which destabilizes its structure and may inﬂ uence complex

interactions that occur in this matrix. As a result of these studies, we are better equipped

to design synthetic collagen for biomedical applications.

16.3.8 Triﬂ uoromethyl - containing Amino Acids in Protein Design

Koksch and co - workers have investigated the effects of replacing a single residue with

several ﬂ uorinated derivatives in the context of a coiled - coil system, where the amino acid

sequence can be described as a heptad repeat. The ﬁ rst and fourth residues ( a and d resi-

dues) of the heptad consist of hydrophobic amino acids. The coiled coil is formed when

two or more strands oligomerize, where the individual strands are α - helices and the strands

wind around each other with a left - handed superhelical twist. Residues at the e and g

positions form interhelical salt - bridges, which can impart parallel or antiparallel speciﬁ city

to the coiled coil. Koksch and co - workers designed a coiled coil to fold in an antiparallel

manner [99] . A single core residue (L9) was replaced by a series of ﬂ uorinated amino

acids and changes in stability were monitored. Experiments were designed to probe the

size of a triﬂ uoromethyl group. The authors posit that if the triﬂ uoromethyl group is as

large as an isopropyl group, triﬂ uoroethyl glycine (TEG) should be as large as leucine. If

this is so, one expects that TEG substitution would not change the stability of the peptide.

In fact, this substitution drastically reduced the stability of coiled coils [99, 100] . However,

a series of additional experiments may be needed in order to ascribe these observations

entirely to size effects as distinct from other parameters, such as packing, dihedral angle

preferences, hydrophobicity, helical propensities, and stereoelectronics.

Substitution of (2 S , 4 S ) - 5 - ﬂ uoroleucine at two positions in ubiquitin decreases its

thermal stability by about 8 ° C [101] . Differential scanning calorimetry revealed a similar

curve for the ﬂ uorinated and wild - type derivatives, where ∆ C

is similar but ∆ H

unf

was

smaller for the ﬂ uorinated derivative. However, numerous studies have been performed

where triﬂ uoromethyl substitutions improve thermal stabilities. In one example, Raleigh

and Horng introduced a single triﬂ uorovaline into the N - terminal domain of ribosomal

protein L9 (NTL9) to investigate its inﬂ uence on the kinetics and thermodynamics of

protein folding [102] . Val3 and Val21 are mostly buried in wild type and occupy positions

in adjacent β - sheets. These residues were separately replaced with triﬂ uorovaline to deliver

two variants tfV3 and tfV21. CD and NMR experiments indicated that both variants folded

into their native states with limited structural perturbation. The folding free energy ( ∆ G ° )

determined by guanidinium hydrochloride (Gdn · HCl) denaturation was 4.17 kcal/mol for

430 Fluorine in Medicinal Chemistry and Chemical Biology

wild type, 4.96 and 5.61 kcal/mol for tfV3 and tfV21, respectively. The elevated stability

by a single CF

group incorporation is quite remarkable and signiﬁ cantly larger than what

has been observed on coiled - coil systems on a per - residue basis.

Our laboratory [103, 104] and that of David Tirrell [105 – 107] have independently

designed protein folds with super thermal and chemical stability. GCN4 - p1, the dimeriza-

tion domain of the yeast transcriptional activator protein bZip, served as a starting point

for the engineering efforts. GCN4 - p1 peptides [108] pack against each other to form a

homodimeric coiled coil with valine in a positions and leucine in every d position. We

envisioned that replacing the core residues with ﬂ uorinated counterparts would increase

the driving force for self - association (Figure 16.8 ). Indeed, incorporation of 4,4,4 - triﬂ uo-

rovaline and 5,5,5 - triﬂ uoroleucine at a and d positions resulted in a coiled coil with higher

stability. The melting temperature ( T

) for the ﬂ uorocarbon peptide was 62 ° C, compared

with 47 ° C for the hydrocarbon peptide. Chaotropic denaturation with Gdn · HCl showed

that the apparent free energy of unfolding of ﬂ uorinated peptides was ∼ 1.0 kcal/mol higher

than the control. The increased thermal and chemical stability could be directly attributed

to the higher hydrophobicity of the CF

over the CH

group [103] .

Tirrell and co - workers substituted all four leucines with triﬂ uoroleucines at the d

position in GCN4 - p1 and observed a similar improvement in thermal stability ( ∆ T

= 13 ° C

at a peptide concentration of 30 µ M) [106] . Next, they set out to ﬂ uorinate the 56 - residue

Figure 16.8 Model GCN4 - p1 peptides. (a) Helical wheel diagram and sequence of GCN4 -

p1 analogue. C

†

= acetamidocysteine. (b) Structures of triﬂ uoroleucine (L) and triﬂ uorovaline

(V) used to stabilize peptide ensembles. The asterisk indicates unresolved stereochemistry.

d positions are shown as spheres. Side - chains of Asn residues are shown in stick representa-

tion. The structure was generated using MacPyMOL (DeLano Scientiﬁ c LLC, Palo Alto, CA,

U.S.A.).