Stabilization of DNA duplex by 2-substituted adenine as a minor groove modifier

2-(1-Naphthalenylethynyl)-2'-deoxyadenosine ((N)A) was synthesized and incorporated into oligodeoxynucleotides. DNA duplexes containing newly designed 5'-(N)AT-3'/3'-T(N)A-5' base pairs are considerably stabilized than unmodified duplexes by stacking interaction of naphthalene rings in the narrow minor groove as characterized by a new emission at longer wavelength and exciton coupled CD signals.     

New reagents and methods for the synthesis of internal and 3'-labeled DNA

The syntheses of two new nucleoside phosphoramidites containing a hydroxyl functionality masked by a levulinate protecting group are presented; N(4)-(2-(ethylene glycol-2-levulinate)ethyl)-5-methyl-5'-(4,4'-dimethoxytrityl)-3'-O-(2-cyanoethyldiisopropylphosphoramidite)-2'-deoxycytidine 1 and 5-(N-(6-O-levulinoyl-1-aminohexyl)-3(E)-acrylamido)-5'-(4,4'-dimethoxytrityl)-3'-(2-cyanoethyldiisopropylphosphoramidite)-2'-deoxyuridine 3. Optimization of solid-phase-supported synthetic parameters for incorporation of these into DNA, removal of the levulinate group by exposure to dilute hydrazine, and subsequent attachment of dye labels is described. Synthesis of the known compound 5-(N-(6-trifluoroacetylaminohexyl)-3(E)-acrylamido)-5'-(4,4'-dimethoxytrityl)-3'-(2-cyanoethyldiisopropylphosphoramidite)-2'-deoxyuridine 2 (1), containing a masked amine at the end of an alkyl chain attached at the 5 position, was also revisited using new techniques developed for 3.     

Measurement of 5-fluoro-2'-deoxyuridine serum levels by gas chromatography chemical ionization mass spectrometry

Serum levels of 5-fluoro-2'-deoxyuridine in cancer treated patients were measured by gas chromatography mass spectrometry under chemical ionization conditions; 1-(2-deoxy-beta-D-lyxofuranosyl)-5-fluorouracil (3'-epi-5-fluoro-2'-deoxyuridine) was used as an internal standard. The drug and internal standard were quantitatively isolated from the serum sample by a mini-column anion exchange method and the extract permethylated using potassium-tert-butoxide in dimethylsulphoxide and methyl iodide. The derivatized nucleosides were then re-extracted from the reaction mixture and analysed on a glass capillary column coated with Superox-4. The column was coupled directly to the chemical ionization source of the mass spectrometer; NH3 was used as the reagent gas. The gas chromatographic effluent was monitored at m/z 289, the [MH]+ ion of the N,O-permethyl derivatives of 5-fluoro-2'-deoxyuridine and the internal standard. Recovery of 5-fluoro-2'-deoxyuridine from serum in the 0-1 microgram ml-1 concentration range averaged 93 +/- 2% (SD); a linear detector response was observed up to 50 ng 5-fluoro-2'-deoxyuridine ml-1. At the 10 ng ml-1 level, a within-run assay precision of 10% (CV) (n = 5) was found, while a detection limit of about 1 ng 5-fluoro-2'-deoxyuridine ml-1 of serum was attained. The method was applied to the measurement of disappearance curves of 5-fluoro-2'-deoxyuridine in the serum of treated patients.     

A bridging water anchors the tethered 5-(3-aminopropyl)-2'-deoxyuridine amine in the DNA major groove proximate to the N+2 C.G base pair: implications for formation of interstrand 5'-GNC-3' cross-links by nitrogen mustards

Site-specific insertion of 5-(3-aminopropyl)-2'-deoxyuridine (Z3dU) and 7-deaza-dG into the Dickerson-Drew dodecamers 5'-d(C (1)G (2)C (3)G (4)A (5)A (6)T (7)T (8)C (9) Z (10)C (11)G (12))-3'.5'-d(C (13)G (14)C (15)G (16)A (17)A (18)T (19)T (20)C (21) Z (22)C (23)G (24))-3' (named DDD (Z10)) and 5'-d(C (1)G (2)C (3)G (4)A (5)A (6)T (7) X (8)C (9) Z (10)C (11)G (12))-3'.5'-d(C (13)G (14)C (15)G (16)A (17)A (18)T (19) X (20)C (21) Z (22)C (23)G (24))-3' (named DDD (2+Z10)) (X = Z3dU; Z = 7-deaza-dG) suggests a mechanism underlying the formation of interstrand N+2 DNA cross-links by nitrogen mustards, e.g., melphalan and mechlorethamine. Analysis of the DDD (2+Z10) duplex reveals that the tethered cations at base pairs A (5).X (20) and X (8).A (17) extend within the major groove in the 3'-direction, toward conserved Mg (2+) binding sites located adjacent to N+2 base pairs C (3).Z (22) and Z (10).C (15). Bridging waters located between the tethered amines and either Z (10) or Z (22) O (6) stabilize the tethered cations and allow interactions with the N + 2 base pairs without DNA bending. Incorporation of 7-deaza-dG into the DDD (2+Z10) duplex weakens but does not eliminate electrostatic interactions between tethered amines and Z (10) O (6) and Z (22) O (6). The results suggest a mechanism by which tethered N7-dG aziridinium ions, the active species involved in formation of interstrand 5'-GNC-3' cross-links by nitrogen mustards, modify the electrostatics of the major groove and position the aziridinium ions proximate to the major groove edge of the N+2 C.G base pair, facilitating interstrand cross-linking.     

Use of monoacetyl-4-hydroxyaminoquinoline 1-oxide to probe contacts between guanines and protein in the minor and major grooves of DNA. Interaction of Escherichia coli integration host factor with its recognition site in the early promoter and transposition enhancer of bacteriophage Mu.

Monoacetyl-4-hydroxyaminoquinoline 1-oxide (Ac-HAQO) reacts with DNA to form adducts at the C8- and N2-positions of guanine and with the N6-position of adenine. Only the N2-guanine adduct blocks the 3'-5' exonuclease action of phage T4 DNA polymerase. Piperidine treatment cleaves the DNA at sites bearing C8-guanine adducts. The N2-position of guanine lies in the minor groove of DNA, whereas the C8-position of guanine occupies the major groove. We have taken advantage of these characteristics to employ Ac-HAQO in conjunction with either T4 DNA polymerase or piperidine in a footprinting technique to probe the interaction of the Escherichia coli integration host factor (IHF) with its binding site. We show that when IHF binds to its recognition site both the N2- and C8-positions of guanines are protected from modification by AcHAQO. In addition, the binding of IHF to DNA was prevented when either an N2- or a C8-AQO adduct was present in the binding site. When dimethylsulfate was used as the footprinting reagent, IHF protected against methylation of the N3 position of adenine in the minor groove but not the N7 position of guanine in the major groove. The difference in results obtained with the two reagents is ascribed to their relative sizes. Both DMS and AcHAQO are excluded by IHF from the minor groove, but only the larger AcHAQO molecule is excluded from the major groove.     

Benzo[a]pyrene-dG adduct interference illuminates the interface of vaccinia topoisomerase with the DNA minor groove

Vaccinia DNA topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a pentapyrimidine target site 5'-C(+5)C(+4)C(+3)T(+2)T(+1)p downward arrow in duplex DNA. The enzyme engages the target site within a C-shaped protein clamp. Here we mapped the interface of topoisomerase with the DNA minor groove by introducing chiral C-10 R and S 7,8-diol 9,10-epoxide adducts of benzo[a]pyrene (BP) at single N(2)-deoxyguanosine (dG) positions within the nonscissile DNA strand. These trans opened BPdG adducts fit into the minor groove without perturbing helix conformation or base pairing, and the R and S diastereomers are oriented in opposite directions within the minor groove. We measured the effects of the BPdG adducts on the rate and extent of single-turnover DNA transesterification. We observed a sharp margin of interference effects, whereby +5 and -2 BPdG modifications were well tolerated but +4, +3, and -1 BPdG adducts were severely deleterious. Stereoselective effects at the -1 nucleoside (the R isomer interfered, whereas the S isomer did not) delineated at high resolution the downstream border of the minor groove interface. BPdG inhibition of transesterification is likely caused by steric exclusion of constituents of the topoisomerase from the minor groove. We also applied the BPdG interference method to probe the interactions of exonuclease III with the minor groove. DNAs containing these BPdG adducts were protected from digestion by exonuclease III, which was consistently arrested at positions 2-4 nucleotides prior to the BP-modified guanosine.     

The N2-guanine adduct but not the C8-guanine or N6-adenine adducts formed by 4-nitroquinoline 1-oxide blocks the 3'-5' exonuclease action of T4 DNA polymerase

When O-acetyl-4-(hydroxyamino)quinoline 1-oxide (Ac-4HAQO) reacts with double-stranded DNA at 37 degrees C the major products, N2-guanine, C8-guanine, and N6-adenine adducts, are formed in the proportions of 5:3:2, respectively. When the reaction is carried out with single-stranded DNA at 0 degree C, the products are found in the ratio 1:7:2. Unique 174-bp DNA fragments were modified in these ways and used as substrates for the 3'-5' exonuclease activity of T4 DNA polymerase. The results obtained showed that the exonuclease is blocked by the N2-guanine adduct but not the other two adducts. Interpretation of the cleavage patterns suggested that the enzyme stopped 2 nucleotides before the N2-guanine adduct. The N2-guanine adduct lies in the minor groove of the DNA double helix, while the other two adducts are found in the major groove. Apparently, only the former hinders progression of the enzyme.     

Sequence-selective targeting of long stretches of the DNA minor groove by a novel dimeric bis-benzimidazole

A dimeric bis-benzimidazole molecule has been designed by computer modeling to bind to a DNA sequence via the DNA minor groove that covers a complete turn of B-DNA. A series of bis-benzimidazole dimers incorporating a -O-(CH(2))(n)()-X-CH(2))(n)()-O- linker, with n = 2 or 3 and X = O or N(+)H(Me), were screened for their capacity to fit the DNA minor groove. The modeling studies enabled an optimal linker to be devised (n = 3, X = N(+)H(Me)), and the synthesis of the predicted "best" molecule, N-methyl-N,N-bis-3,3-[4'-[5' '-(2' "-p-methoxyphenyl)-5' "-1H-benzimidazolyl]-2' '-1H-benzimidazolyl]phenoxypropylamine (5), is reported. The optimized linker permits the two symmetric bis-benzimidazole motifs to maintain hydrogen-bonded contacts with the floor of the DNA minor groove. DNase I footprinting studies have shown that this ligand binds with high affinity to sequences representing approximately a complete turn of B-DNA, represented by the [A.T](4)-[G.C]-[A.T](4) motif, and only poorly to sequences of half this site size, in accord with the computer modeling studies. Compound 5 does not show acute cellular cytotoxicity, in contrast with its monomeric bis-benzimidazole precursors, yet is rapidly taken up into cells.     

Synthesis of N,N-dialkylaniline-2'-deoxyuridine conjugates for DNA-mediated electron transfer studies

Syntheses of two analogs of deoxyuridine with N,N-dialkylaniline chromophores are reported. 5-[3-(N-methylphenylamino)propanoyl]-2'-deoxyuridine (1) and 5-[2-(4-N,N-dimethylaminophenyl)ethyl)]-2'-deoxyuridine (2) are prepared by palladium-mediated coupling. Preparation of 2 was facilitated by in situ transient O4-trimethylsilyl protection during alkynylation which suppressed secondary cyclization of the coupling adduct.     

Antisense oligodeoxynucleotides: synthesis, biophysical and biological evaluation of oligodeoxynucleotides containing modified pyrimidines.

6-Azathymidine, 6-aza-2'-deoxycytidine, 6-methyl-2'-deoxyuridine, and 5,6-dimethyl-2'-deoxyuridine nucleosides have been converted to phosphoramidite synthons and incorporated into oligodeoxynucleotides (ODNs). ODNs containing from 1 to 5 of these modified pyrimidines were compared with known 2'-deoxyuridine, 5-iodo-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, 5-fluoro-2'-deoxyuridine, 5-bromo-2'-deoxycytidine, and 5-methyl-2'-deoxycytidine nucleoside modifications. Stability in 10% heat inactivated fetal calf serum, binding affinities to RNA and DNA complements, and ability to support RNase H degradation of targeted RNA in DNA-RNA heteroduplexes were measured to determine structure-activity relationships. 6-Azathymidine capped ODNs show an enhanced stability in serum (7- to 12-fold increase over unmodified ODN) while maintaining hybridization properties similar to the unmodified ODNs. A 22-mer ODN having its eight thymine bases replaced by eight 6-azathymines or 5-bromouracils hybridized to a target RNA and did not inhibit RNase H mediated degradation.     

DNA determinants important in sequence recognition by Eco RI endonuclease

Alkylation interference and protection methods (Siebenlist, U., and Gilbert, W., (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 122-126) have been utilized to deduce potential DNA contacts involved in specific complex formation between Eco RI endonuclease and its recognition sequence. The endonuclease protected the N7 position (major groove) of the dG and the N3 position (minor groove) of both dA residues within the Eco RI sequence against alkylation by dimethylsulfate, d(GpApApTpTpC), suggesting the presence of poly-peptide in both grooves in the vicinity of affected nitrogens. Results of methylation interference analysis suggest that the N7 of the Eco RI site dG and the N3 of the central dA, d(GpApApTpTpC), are utilized as contacts by the enzyme. The failure to observe interference upon methylation of the 5'-penultimate dA within the sequence implies that the endonuclease does not bond to the N3 of this residue, despite the fact that it is protected against alkylation by the protein. Ethylation interference patterns suggest four major phosphate contacts between endonuclease and each DNA strand. Two of these phosphates are 5'-external to the Eco RI sequence, d(pNpGpApApTpTpC), suggesting involvement of outside phosphates in electrostatic interactions. Moreover, alkylation protection and interference effects on the two DNA strands display perfect 2-fold symmetry. Thus, the endonuclease interacts with a minimum of 10 nucleotide pairs to yield a DNA-protein complex characterized by elements of symmetry. In contrast, specific alkylation effects were not observed in comparable experiments with the endonuclease and a DNA which had been previously methylated by the Eco RI modification enzyme.     

Structure of a beta-alanine-linked polyamide bound to a full helical turn of purine tract DNA in the 1:1 motif

Polyamides composed of N-methylpyrrole (Py), N-methylimidazole (Im) and N-methylhydroxypyrrole (Hp) amino acids linked by beta-alanine (beta) bind the minor groove of DNA in 1:1 and 2:1 ligand to DNA stoichiometries. Although the energetics and structure of the 2:1 complex has been explored extensively, there is remarkably less understood about 1:1 recognition beyond the initial studies on netropsin and distamycin. We present here the 1:1 solution structure of ImPy-beta-Im-beta-ImPy-beta-Dp bound in a single orientation to its match site within the DNA duplex 5'-CCAAAGAGAAGCG-3'.5'-CGCTTCTCTTTGG-3' (match site in bold), as determined by 2D (1)H NMR methods. The representative ensemble of 12 conformers has no distance constraint violations greater than 0.13 A and a pairwise RMSD over the binding site of 0.80 A. Intermolecular NOEs place the polyamide deep inside the minor groove, and oriented N-C with the 3'-5' direction of the purine-rich strand. Analysis of the high-resolution structure reveals the ligand bound 1:1 completely within the minor groove for a full turn of the DNA helix. The DNA is B-form (average rise=3.3 A, twist=38 degrees ) with a narrow minor groove closing down to 3.0-4.5 A in the binding site. The ligand and DNA are aligned in register, with each polyamide NH group forming bifurcated hydrogen bonds of similar length to purine N3 and pyrimidine O2 atoms on the floor of the minor groove. Each imidazole group is hydrogen bonded via its N3 atom to its proximal guanine's exocyclic amino group. The important roles of beta-alanine and imidazole for 1:1 binding are discussed.     

Base-boronated dinucleotides: synthesis and effect of N7-cyanoborane substitution on the base protons.

Boron-modified nucleic acids comprise a new set of DNA mimics that have potential biological and therapeutic applications. A series of nine dinucleotides containing N7-cyanoborane-2'-deoxyguanosine ((7b)dG) at the 3', 5' or both positions of the phosphodiester linkage have been synthesized using solution phase phosphoramidite chemistry. Fmoc was used as the 5'-protecting group because of incompatibility of the cyanoborane moiety with 5'-DMT cations generated during the deprotection step. The presence of the cyanoborane group was confirmed on the basis of Fab-MS and 1H NMR spectroscopy. The H-8 proton of (7b)dG in the dinucleotides shifted 0.35-0.80 p.p.m. downfield relative to that of unmodified dG. A comparison of the D20 exchange kinetics of the H-8 proton at 60 degrees C showed that H-8 of (7b)dG is very labile relative to unmodified dG, indicating that the N7-cyanoborane modification increases the acidity of the H-8 proton of (7b)dG. These studies illustrate the feasibility of synthesizing boron-containing oligonucleotides which are modified at the N7-guanine to block Hoogsteen pairing in the DNA major groove.     

Hydrogen and hydration of DNA and RNA oligonucleotides

In this paper, hydrogen bonding interaction and hydration in crystal structures of both DNA and RNA oligonucleotides are discussed. Their roles in the formation and stabilization of oligonucleotides have been covered. Details of the Watson-Crick base pairs G.C and A.U in DNA and RNA are illustrated. The geometry of the wobble (mismatched) G.U base pairs and the cis and almost trans conformations of the mismatched U.U base pairs in RNA is described. The difference in hydration of the Watson-Crick base pairs G.C, A.U and the wobble G.U in different sequences of codon-anticodon interaction in double helical molecules are indicative of the effect of hydration. The hydration patterns of the phosphate, the 2'-hydroxyl groups, the water bridges linking the phosphate group, N7 (purine) and N4 of Cs or O4 of Us in the major groove, the water bridges between the 2'-hydroxyl group and N3 (purine) and O2 (pyrimidine) in the minor groove are discussed.     

Expression of herpes virus thymidine kinase in Neurospora crassa.

The expression of thymidine kinase in fungi, which normally lack this enzyme, will greatly aid the study of DNA metabolism and provide useful drug-sensitive phenotypes. The herpes simplex virus type-1 thymidine kinase gene ( tk ) was expressed in Neurospora crassa. tk was expressed as a fusion to N.crassa arg-2 regulatory sequences and as a hygromycin phosphotransferase-thymidine kinase fusion gene under the control of cytomegalovirus and SV40 sequences. Only strains containing tk showed thymidine kinase enzyme activity. In strains containing the arg-2 - tk gene, both the level of enzyme activity and the level of mRNA were reduced by growth in arginine medium, consistent with control through arg-2 regulatory sequences. Expression of thymidine kinase in N.crassa facilitated radioactive labeling of replicating DNA following addition of [3H]thymidine or [14C]thymidine to the growth medium. Thymidine labeling of DNA enabled demonstration that hydroxyurea can be used to block replication and synchronize the N.crassa mitotic cycle. Strains expressing thymidine kinase were also more sensitive than strains lacking thymidine kinase to anticancer and antiviral nucleoside drugs that are activated by thymidine kinase, including 5-fluoro-2'-deoxyuridine, 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouridine and trifluorothymidine. Finally, expression of thymidine kinase in N. crassa enabled incorporation of bromodeoxyuridine into DNA at levels sufficient to separate newly replicated DNA from old DNA using equilibrium centrifugation.     

Antiviral activity of some 5-arylethynyl 2'-deoxyuridine derivatives

5-(3-Perylenylethynyl)-2'-deoxyuridine was prepared by cross-linking 5-iodo-2'-deoxyuridine derivatives with 3-ethynylperylene followed by deprotection. 5-(1-Perylenylethynyl)-, 5-(3-perylenylethynyl)-, and 5-[4-(2-benzoxazolyl)phenylethynyl]-2'-deoxyuridine were found to inhibit in Vero cells the replication of type 1 herpes simplex virus and its drug-resistant strains.     

Intercalating polycyclic aromatic hydrocarbon-DNA adducts poison DNA religation by Vaccinia topoisomerase and act as roadblocks to digestion by exonuclease III

Polycyclic aromatic hydrocarbon (PAH)-DNA adducts pervert the execution or fidelity of enzymatic DNA transactions and cause mutations and cancer. Here, we examine the effects of intercalating PAH-DNA adducts on the religation reaction of vaccinia DNA topoisomerase, a prototypal type IB topoisomerase (TopIB), and the 3' end-resection reaction of Escherichia coli exonuclease III (ExoIII), a DNA repair enzyme. Vaccinia TopIB forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a target site 5'-C(+5)C(+4)C(+3)T(+2)T(+1)p / N(-1) in duplex DNA. The rate of the forward cleavage reaction is suppressed to varying degrees by benzo[a]pyrene (BP) or benzo[c]phenanthrene (BPh) adducts at purine bases within the 3'-G(+5)G(+4)G(+3)A(+2)A(+1)T(-1)A(-2) sequence of the nonscissile strand. We report that BP adducts at the +1 and -2 N6-deoxyadenosine (dA) positions flanking the scissile phosphodiester slow the rate of DNA religation to a greater degree than they do the cleavage rate. By increasing the cleavage equilibrium constant > or = 10-fold, the BPdA adducts, which are intercalated via the major groove, act as TopIB poisons. With respect to ExoIII, we find that (i) single BPdA adducts act as durable roadblocks to ExoIII digestion, which is halted at sites 1 and 2 nucleotides prior to the modified base; (ii) single BPhdA adducts, which also intercalate via the major groove, elicit a transient pause prior to the lesion, which is eventually resected; and (iii) BPh adducts at N2-deoxyguanosine, which intercalate via the minor groove, are durable impediments to ExoIII digestion. These results highlight the sensitivity of repair outcomes to the structure of the PAH ring system and whether intercalation occurs via the major or minor groove.     

Antiviral Activity of Some 2'-Deoxyuridine 5-Arylethynyl Derivatives

5-(3-Perylenylethynyl)-2'-deoxyuridine was prepared by crosslinking 5-iodo-2'-deoxyuridine derivatives with 3-ethynylperylene followed by deprotection. 5-(1-Perylenylethynyl)-, 5-(3-perylenylethynyl)-, and 5-[4-(2-benzoxazolyl)phenylethynyl]-2'-deoxyuridine were found to inhibit in Vero cells the replication of type 1 herpes simplex virus and its drug-resistant strains.     

Synthesis, conversion and biological activity of 5-substituted 2'-deoxyuridines modified by carbohydrates]

5-Benzyloxymethyl(Bom)-2'-deoxyuridine and its alpha-anomer were used as the key compounds for syntheses of thymidine analogues or 3'-derivatives. Anomeric 5-Bom-2'-deoxyuridines were synthesized from 5-Bom-uracil and 2-deoxy-3,5-di-O-p-toluyl-alpha-D-ribo-furanosyl chloride by means of the silyl method. 5-Bom-2'-deoxyuridine was transformed successively to 3',5'-di-O-mesyl derivative, 2,3'-anhydro-1-(2-deoxy-5-O-p-toluyl-beta-D-xylofuranosyl)-5-Bom-uracil and 3'-azido-2',3'-dideoxy-5-Bom-uridine. Treatment of the last with SnCl4 in methylene dichloride--methanol led to 3'-azido-2',3'-dideoxy-5-methoxymethyluridine. Under the same conditions the 5-methoxymethyl derivative was obtained from 3',5'-di-O-p-toluyl-5-Bom-2'-deoxyuridine. Interaction of 1-(2-deoxy-alpha-D-ribofuranosyl)-4-Bom-uracil with SnCl4 in methylene dichloride as well as the hydrogen transfer hydrogenolysis in the presence of cyclohexene and Pd(OH)2/C in ethanol led to 1-(2-deoxy-alpha-D-ribofuranosyl)-5-hydroxymethyluracil. Only 3'-azido-2',3'-dideoxy-5-Bom-uridine showed a cytotoxic activity against CaOv cells in vitro: in 10(-5)-10(-4) M concentrations it inhibits the thymidine incorporation into DNA by 78.8-95.1%. Elucidation of antitumor activity in vivo showed that this nucleoside inhibits growth of solid tumours, Ca755 and LLC, by 79 and 79-83%, respectively, but has no therapeutic effect against lympholeukemia P388.     

Tuning mesomorphic properties and handedness of chiral calamitic liquid crystals by minimal modification of the effective core

Two pairs of calamitic liquid crystalline molecules, (+)-2-[4'-(S)-sec-butoxyphenyl]-5-(4'-hexoxyphenyl)toluene ((+)-S-1) and (+)-2-(4'-hexoxyphenyl)-5-[4'-(S)-sec-butoxyphenyl]toluene ((+)-S-2), (-)-2-[4'-(R)-sec-butoxyphenyl]-5-(4'-hexoxyphenyl)toluene ((-)-R-1) and (-)-2-(4'-hexoxyphenyl)-5-[4'-(R)-sec-butoxyphenyl]toluene ((-)-R-2), have been designed and synthesized. Each of the molecules consists of a p-terphenyl core substituted with a methyl group on the middle ring, a chiral sec-butoxy tail, and an achiral n-hexoxy tail. The geometrical difference between (+)-S-1 ((-)-R-1) and (+)-S-2 ((-)-R-2) lies only in the location of the methyl group on the effective mesogenic core. Yet, such a small change in the structure gives rise to remarkable differences in mesogenic properties and handedness. Both (+)-S-1 and (-)-R-1 have an enantiotropic cholesteric phase (N*) and a monotropic twist grain boundary C* phase (TGBC*), whereas (+)-S-2 and (-)-R-2 exhibit only a monotropic N* phase. Moreover, (+)-S-1 ((-)-R-1) and (+)-S-2 ((-)-R-2) have opposite handedness in the N* phase, and (+)-S-1 and (-)-R-1 even have a helical inversion from N* to TGBC* phase through a non-helical chiral mesophase.