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

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Fluorinated Moieties for Replacement of Amide and Peptide Bonds 277

sponding PNA sequence, which was equipped with lysine units at the N - termini to enhance

water solubility and for easy puriﬁ cation.

The T

value of ﬂ uoroalkene isostere 89a with antiparallel DNA component was

found to be slightly higher than that of the normal PNA oligomer 88 ( ∆ T

= +2.4 ° C) (see

Figure 10.8 ). This result indicates better stabilization of the PNA/DNA duplex through

introduction of a ﬂ uoroalkene unit at the central part of the PNA backbone. Nonﬂ uorinated

( E ) - alkene isostere 90 [ note: The ( E ) - alkene isostere has the same geometric conﬁ guration

as the ( Z ) - ﬂ uoroalkene isostere] at the same location in the PNA sequence provided better

stabilization of the PNA/DNA duplex with antiparallel DNA component than that of 88

( ∆ T

= +3.5 ° C). In contrast, nonﬂ uorinated ( Z ) - alkene isostere 91 resulted in signiﬁ cant

destabilization of the duplex ( ∆ T

= − 5.2 ° C). Thus, it is suggested that the Z - conﬁ guration

of the double bond may cause a structural mismatch. Interestingly, the stability of the

PNA/DNA duplexes of decamers 89 , containing single ﬂ uoroalkene modiﬁ cation with

antiparallel DNA, was found to be strongly dependent on its position in the PNA sequence,

with ∆ T

values ranging from +2.4 to − 8.1 ° C. Additionally, a similar tendency was

observed for the PNA/DNA duplexes with parallel DNA.

10.2.2.4 Fluoroarenes as Nucleoside Base Mimics

The use of ﬂ uoroarene derivatives as structural mimetics of nucleobases has been studied

extensively [47, 48] . In pioneering work in this ﬁ eld, Kool and co - workers reported the

structural similarity between natural 2 ′ - deoxythymidine (dT) and 1 ′ ,2 ′ - dideoxy - 1 ′ - (2,4 -

diﬂ uoro - 5 - methylphenyl) - β - d - ribofuranoside 92 (dF) (see Figure 10.9 ) [49, 50] .

As shown in Scheme 10.27 , ﬂ uoroarene isostere 92 was prepared from 2,4 - diﬂ uoro -

5 - bromotoluene and silyl - protected lactone 93 in three steps [51] . Thus, the coupling

reaction of lactone 93 with an aryl Grignard regent, which was prepared from 2,4 - diﬂ uoro -

5 - bromotoluene and magnesium in the presence of catalytic amount of iodine, and the

subsequent reductive dehydroxylation of lactol intermediate by Et

SiH resulted in the β -

selective introduction of diﬂ uorotoluyl group. Further treatment with TBAF deprotected

the silyl groups to give 92 in excellent yield. With the use of essentially the same proce-

dure, dichloro - and dibromo - analogues were also prepared. To incorporate 92 into the

DNA sequence, 5 ′ - OH selective protection of 92 with 4,4 ′ - dimethoxytrityl chloride (DMTr -

Cl) and subsequent phosphorylation with 2 - cyanoethyl N , N - diisopropylchlorophosphora-

midite gave the fully protected nucleotide precursor 94 , which was subjected to

Figure 10.8 T

data of PNA, alkenic PNA and ﬂ uoroalkenic PNA.

278 Fluorine in Medicinal Chemistry and Chemical Biology

Figure 10.9 Natural 2 ′ - deoxythymidine and 1 ′ ,2 ′ - dideoxy - 1 ′ - (2,4 - diﬂ uoro - 5 - methylphenyl) - β - D -

ribofuranoside.

Scheme 10.27

oligonucleotide synthesis using an automated synthesizer based on phosphoramidite chem-

istry [52] .

Fluoroarene isostere 92 was designed as a nonpolar nucleoside with the closest pos-

sible steric and structural similarity to the natural deoxythymidine, but without hydrogen -

bonding ability. Despite the proposed ability of C – F groups in ﬂ uoroarene derivatives as

weak hydrogen - bonding acceptors and aromatic C – H groups as weak hydrogen - bonding

donors [53, 54] ,

H - NMR titration of 9 - ethyladenine with 2,4 - diﬂ uorotoluene in aqueous

media did not show signiﬁ cant change of chemical shifts in 9 - ethyladenine [50] . In general,

it is known that the hydrogen - bonds between molecules in aqueous solution are weak due

to the high dielectric constant of the medium as well as competitive hydrogen - bonding

with the solvent itself. Therefore, this observation indicates the lack of hydrogen - bonding

between the ﬂ uoroarene nucleoside isostere and the complementary natural nucleoside in

aqueous media. On the other hand, Engeles and co - workers recently found intermolecular

C – H · · · F – C hydrogen bonding in the crystalline form of 1 ′ - deoxy - 1 ′ - (4 - ﬂ uorophenyl) - β - d -

ribofuranose 95a (see Figure 10.10 ) [55] . The x - ray crystallographic analysis of 95a

(recrystallized from methanol) conﬁ rmed that the distance between the ﬂ uorine atom and

the arylic hydrogen at the 3 - position of another molecule (2.30 Å ) is signiﬁ cantly shorter

than the sum of the van der Waals radii of ﬂ uorine and hydrogen (2.55 Å ). Unfortunately,

Fluorinated Moieties for Replacement of Amide and Peptide Bonds 279

in the cases of 3 - ﬂ uorophenyl - β - d - ribofuranose 95b and 2,4 - diﬂ uorophenyl - β - d - ribofura-

nose 95c , no H · · · F distance shorter than 2.55 Å was observed. This intermolecular C –

H · · · F – C hydrogen bond also showed nearly linear arrangement with dihedral angle of

158 ° . Interestingly, the H · · · F distance in a single crystal of 95a recrystallized from water

was found to be longer (2.38 Å ) than in that recrystallized from methanol (2.30 Å ) due to

the incorporation of a water molecule into the unit cell: that is, a water molecule is placed

between 2 ′ - OH and 5 ′ - OH, which makes the F · · · H distance longer.

Engeles and co - workers also reported the effect of these ﬂ uoroarene isosteres on the

thermodynamic properties of the duplex RNA [55] (see Figure 10.11 ). In the 12 - mer oli-

gonucleotides (5 ′ - CUU UUC XUU CUU paired with 3 ′ - GAA AAG YAA GAA), the

Figure 10.10 Fluoroarene nucleoside isosteres.

Figure 10.11 Thermodynamic properties of modiﬁ ed/unmodiﬁ ed duplex RNA (5 ′ - CUU

UUC XUU CUU paired with 3 ′ - GAA AAG YAA GAA).

280 Fluorine in Medicinal Chemistry and Chemical Biology

wobble base pair U · G showed the highest T

(38.6 ° C). Single modiﬁ cation of oligonucle-

otide sequence by the incorporation of nonﬂ uorinated phenyl isostere 95d signiﬁ cantly

destabilized the RNA duplex structure, although ﬂ uorinated analogues 95a , 95b and 95c

improved the thermal stability of the duplex RNA compared with 95d . In particular, the

values of singly modiﬁ ed duplex RNAs with 2,4 - diﬂ uorophenyl isostere 95c were

notably higher than those with monoﬂ uorinated isosteres. 4,6 - Diﬂ uorobenzoimidazole

isostere 96 was also found to be an effective mimic of purine bases such as inosine. Sur-

prisingly, the pairing of ﬂ uorinated nucleobase isosteres with a purine or pyrimidine

nucleobase resulted in similar T

values. Thus, these ﬂ uoroarene isosteres potentially act

as a new class of universal base, which can pair with all natural bases without energy loss.

The evaluation of doubly modiﬁ ed duplex RNAs also suggested a signiﬁ cant interaction

between 96 and 95c ( T

= 34.6 ° C). It was also demonstrated by the exhaustive thermal

analysis that the duplex RNA with 95c · 96 base pair is 0.6 kcal/mol more stable than that

of the calculated value. To explain this stabilization, Engeles and co - workers proposed a

weak F · · · H hydrogen - bond between the ﬂ uorinated nucleotides [55] .

Kool and co - workers also reported 1 ′ ,2 ′ - dideoxy - 1 ′ - (4 - ﬂ uoro - 6 - methylbenzoimid-

azolyl) - β - d - ribofuranose ( 97 , dH) as a highly effective nonpolar isostere of 2 ′ - deoxy-

guanosine (dG) (see Figure 10.12 ) [56] . In crystalline forms, the bond lengths and base

shapes in these compounds are quite close, although the orientation of the base with respect

to the furanose ring is different. That is, the natural dG adopts an anti conformation with

a torsion angle χ of − 122 ° , while the base part of 97 is twisted by 58 ° relative to this and

falls between typical anti and syn ranges with a measured torsion angle χ of − 65 ° .

However, NMR studies in D

O revealed that the conformation of 97 in solution was

essentially identical to that of dG. For instance, the conformations of the deoxyribose parts

of both nucleosides were classiﬁ ed to be 70% S for dG and 66% S for 97 . In general, the

2 - deoxyribofuranoside structure preferentially occupies the S (2 ′ - endo ) conformation.

Additionally, nuclear Overhauser effect (NOE) data of 97 in D

O strongly indicate that

nucleoside 97 is favored as the anti - conformer.

One of the most important aspects of 4 - ﬂ uoro - 6 - methylbenzimidazole as a modiﬁ ed

nucleobase is its strong stabilization of duplex DNA through a stacking effect. The stack-

ing properties of 97 and other modiﬁ ed nucleosides can be measured by the dangling end

thermal denaturation studies of DNA duplexes with self - complementary stands (dXCGC-

Figure 10.12 2 ′ - Deoxyguanosine and 1 ′ ,2 ′ - dideoxy - 1 ′ - (4 - ﬂ uoro - 6 - methylbenzoimidazolyl) - β - D - ribo -

furanose.

Fluorinated Moieties for Replacement of Amide and Peptide Bonds 281

GCG.) (see Figure 10.13 a) [57] . Regarding the free energy of stacking for the natural/

modiﬁ ed nucleobases, this study showed that the nucleobase mimic 97 stacked consider-

ably more strongly than any of the four natural DNA bases. A possible stacking model

was proposed as shown in Figure 10.13 . In this model, the 4 - ﬂ uoro - 6 - methylbenzimid-

azole ring nicely overlaps the cytosine base.

As an unique application of ﬂ uoroarene isosteres, Kool and co - workers recently

reported that sets of 1 ′ ,2 ′ - dideoxy - 1 ′ - (2,4 - dihalo - 5 - methylphenyl) - β - d - ribofuranose ( 98 )

and 1 ′ - deoxy - 1 ′ - (2,4 - dihalo - 5 - methylphenyl) - β - d - ribofuranose ( 99 ) as thymidine or

uridine analogues with gradually increasing size can be used as “ molecular rulers ” (see

Figure 10.14 ) [56] . X - ray crystallographic analysis and various NMR studies in D

showed that the conformations of these isosteres in both crystalline state and solution

closely resemble that of thymidine. Additionally, PM3 calculations of 98 and 99 suggested

that change of the substituent X from hydrogen to a series of halogen atoms would increase

the bond lengths at the 2,4 - positions, corresponding to oxygens of thymidine, ranging from

1.09 Å to 1.97 Å (see Figure 10.14 ). Therefore, enzymatic evaluation using a set of these

compounds should be able to elucidate the ﬁ delity of substrate speciﬁ city for the target

enzyme.

According to this concept, in 2005 Kool and Essingmann reported the utility of

“ molecular rulers ” as probes for the active - site tightness of DNA polymerase [58, 59] . In

kinetic studies of single nucleotide insertion in the steady state using a gel electorophore-

sis - based analysis, it was found that the efﬁ ciency of the replication of 28 - mer/23 - mer

template - primer duplexes, having the sequence (5 ′ - ACT G A T CTC CCT ATA GTG AGT

CGT ATT A · 5 ′ - T AAT ACG ACT CAC TAT AGG GAG A) with variably sized nucleoside

triphosphates 100 promoted by DNA polymerase I from E. coli (Klenow fragment, exo-

nuclease deﬁ cient), was highly dependent on the size of compounds (see Figure 10.15 ).

A comparison of the efﬁ ciency for nucleotide insertions, which was measured by V

max

/ K

shows that the Cl - substituted analogue dLTP, whose chloroarene ring is larger than natural

thymine by 0.5 Å , is the most efﬁ cient compound in this series. In addition, the relative

efﬁ ciency of dLTP is almost the same as that of natural dTTP within experimental error,

Figure 10.13 (a) The helix – coil equilibrium of 5 ′ - XCGCGCG and dangling end thermal

denaturation studies. (b) Possible 5 ′ - end stacking geometry for the aromatic rings.

282 Fluorine in Medicinal Chemistry and Chemical Biology

Figure 10.14 PM3 - calculated bond length of arene isosteres.

Figure 10.15 Steady - state kinetic efﬁ ciencies for single - nucleotide insertion by DNA poly-

merase I with variably sized nucleoside triphosphate.

despite the nonpolar nature of dLTP. When the molecule size exceeded that of the chlori-

nated analogue, the relative efﬁ ciency dropped markedly by a factor of 164 for the largest

dITP in response to a subtle size increase of 0.35 Å over the optimum. This result suggests

that the active site of DNA polymerase strictly recognizes the steric bulkiness and the

shape of the substrate.

The “ molecular ruler ” methodology for determining the size of the active site of DNA

polymerase was also applied to the investigations of other DNA polymerases, such as T7

DNA polymerase and Dpo4 polymerase [60, 61] .

On the other hand, a set of 1 ′ - deoxy - 1 ′ - (2,4 - dihalo - 5 - methylphenyl) - β - d - ribofura-

noses ( 99 , rN) was used for the investigation of ﬂ exible active site of reverse transcriptase

of HIV - 1 virus (HIV - RT) (see Figure 10.16 ) [62] . Since HIV - RT is well known as a highly

mutagenic polymerase, the understanding of its mutagenicity potentially provides a solu-

tion to the rapid development of drug resistance during treatment of AIDS. The efﬁ ciencies

of the dATP incorporation to singly modiﬁ ed RNA templates by HIV - RT are shown in

Figure 10.16 . All uracil analogues 99 directed preferential incorporation of dATP. Regard-

Fluorinated Moieties for Replacement of Amide and Peptide Bonds 283

ing the effect of size on the efﬁ ciency, the smallest analogue (X = H) was less tolerated

than other analogues in the template, but there was otherwise little or no difference in

dATP incorporation efﬁ ciencies. The results are remarkably different from those for the

DNA polymerases, and clearly suggest the high structural ﬂ exibility of HIV - RT.

10.3 Triﬂ uoromethylated Alkenes Ψ [CH(CF

) = CH] as Dipeptide Isosteres

In 1998, Wipf and co - workers demonstrated that the replacement of a peptidic amide bond

with a triﬂ uoromethylated alkene mimics well the β - turn structure of the original peptide

[7] . AM1 - calculated dipolar moments of the amide bond and various alkene isosteres sug-

gested the electrostatic similarity between the amide and the triﬂ uoromethylalkene func-

tionalities (see Figure 10.17 ) [7] .

10.3.1 Synthetic Method

The synthesis of triﬂ uoromethylalkene dipeptide isosteres was reported by Wipf and co -

workers [7] . As shown in Scheme 10.28 , the multistep synthesis of Ala - Ψ [C(CF

) = CH] -

Xaa type isosteres was achieved from 1,1,1 - trichloro - 2,2,2 - triﬂ uoroethane. The

carboxylation of triﬂ uorotrichloroethane via triﬂ uorodichloroethylzinc intermediate, ester-

iﬁ cation with benzyl alcohol, and Reformatsky addition – elimination with acetaldehyde

Figure 10.16 Steady - state kinetic efﬁ ciencies for single - nucleotide insertion by HIV - 1 reverse

transcriptase to duplex RNA templates [5 ′ - r(ACU GXU CUC CCU AUA GUG AGU CGU

AUU A), · 55 ′ - d(T AAT ACG ACT CAC TAT AGG GAG A)] with dATP.

Figure 10.17 Dipolar moments (debye) of amide and various alkene isosteres.

284 Fluorine in Medicinal Chemistry and Chemical Biology

yielded α - triﬂ uoromethylated crotonate 101 as a 2 : 3 mixture of E - and Z - isomers. The

Sharpless asymmetric dihydroxylation of ( Z ) - 101 , which was isolated by column chroma-

tography on silica gel, afforded chiral diol in 73% ee. Conversion of this diol to an epoxide

under Mitsunobu conditions, half - reduction of the ester moiety with DIBAL - H, and Wittig

oleﬁ nation gave triﬂ uoromethylated epoxide 102 . Introduction of amino group through

ring opening of epoxide 102 , followed by allylic methylation of the resultant γ - mesyloxy -

α , β - enoate 103 with methylcuprate, and the ﬁ nal amidation gave Ala - Ψ [C(CF

) = CH] - Ala

104 .

Later, Wipf and co - workers also reported the improved synthesis of triﬂ uoromethyl-

alkene isostere 110 through addition reaction of internal alkynes to N - phosphonylimines

(see Scheme 10.29 ) [63] . As shown in Scheme 10.29 , the reaction of N - phosphonylimine

104 with vinylzinc species, derived from alkynylstannane 103 through hydrozirconation,

followed by transmetallation with Me

Zn, gave vinylstannane 105 in 65% yield. The reac-

tion of 105 with NIS ( N - iodosuccinimide) afforded iodoalkene 106 in 63% yield. It should

be noted that vinylstannane 105 was also converted to ﬂ uoroalkene 107 in moderate yield

through reaction with XeF

and AgOTf [64] . The resulting iodoalkene 106 was trans-

formed to naphthylamide 109 in a further four steps. Finally, the stereoselective introduc-

tion of a CF

group through reaction of 109 with FSO

Me [65] in the presence of

Cu(I) thiophenecarboxylate (CuTc) gave triﬂ uoromethylalkene isostere 110 in reasonable

yield.

10.3.2 Conformational Analysis and Biological Aspects

One of the most important structural properties of triﬂ uoromethylalkene isosteres is the

reproduction of β - turn topography. As shown in Figure 10.18 , natural β - turn structure can

Scheme 10.28

Fluorinated Moieties for Replacement of Amide and Peptide Bonds 285

Scheme 10.29

Figure 10.18 Structure and classiﬁ cation of β - turns. Type IV β - turns are deﬁ ned as those

having two or more angles which differ by at least 40 ° from the deﬁ nitions of β - turn types I,

I ′ , II, II ′ , III and III ′ . Type VI β - turns have a cis - Pro at the position i +2. Type VII β - turns form

a kink in the protein chain created by ψ

≈ 180 ° and | φ

| < 60 ° or | ψ

| < 60 ° and

≈ 180 ° .

be classiﬁ ed broadly into 11 types [66] . Compared with nonﬂ uorinated alkene and meth-

ylalkene isosteres, the corresponding triﬂ uoromethylalkene isosteres act as more effective

β - turn promoters. Thus, the combination of A

1,3

and A

1,2

strains at the triﬂ uoromethylated

alkyne moiety leads to considerable restrictions in φ , ψ - dihedral angles in these peptides.

286 Fluorine in Medicinal Chemistry and Chemical Biology

For instance, BocNH - l - Ala - Ψ [C(CF

) = CH] - d - Ala - CONHMe takes solely type - II β -

turn structure ( φ

= − 60 ° , ψ

= 120 ° , φ

= 110 ° , ψ

= − 23 ° ) in crystalline form (see Figure

10.19 ). In this case, the corresponding methylated alkene isostere also exists as a similar

conformer in crystalline form, although the simple alkene isostere does not have a hydro-

gen - bond between the carbonyl oxygen at residue 1 and the amide proton at residue 4.

A similar observation was reported for the triﬂ uoromethylalkene isostere of gramici-

din S (GS), which is an antibiotic cyclodecapeptide (see Figure 10.20 ) [67] . In natural GS,

the rigid and amphipathic antiparallel β - pleated sheet is held in place by two type - II ′ β -

turns at both d - p he - Pro positions as well as four intramolecular hydrogen - bonds between

the valine and leucine residues. Base on the x - ray crystallographic analysis, it is conﬁ rmed

that triﬂ uoromethylalkene isostere 102 adopts the pleated antiparallel β - sheet structure

with two hydrogen bonds in solid state. The d - p he - Pro unit has an ideal set of dihedral

angles for the type II ′ β - turn ( φ

= 137 ° , ψ

= − 95 ° , φ

= − 82 ° , ψ

= − 5.7 ° ). Comparison

of natural GS and Ψ [C(CF

) = CH]

GS in solution by variable temperature NMR and CD

(circular dischroism) analyses also indicates the structural similarity of these two com-

pounds. In contrast, CD spectra of nonﬂ uorinated isostere Ψ [C(CH

) = CH]

GS 103 suggest

the presence of disordered peptide conformation. Antibacterial activities of des - Cbz - 102

and des - Cbz - 103 also mimic that of the natural GS (MIC = 5 – 15 µ g/mL).

Figure 10.19 β - Turn structure of BocNH - L - Ala - ψ [C(CF

) = CH] - D - Ala - CONHMe.

Figure 10.20 Structures of gramicidin S, Ψ [C(CF

) = CH]

GS, and Ψ [C(CH

) = CH]

GS.