JP2007314477A - Hair-pin polyamide and its constituent molecule - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a substituent of a polyamide having a DNA base recognition power and flexibility to fit its orientation structure with the curvature of the DNA. <P>SOLUTION: The substituent is preferably an α-substituted γ-aminoalkylcarboxylic acid group, especially α-substituted γ-aminobutyric acid. Hydroxyl group or amino group can be suitably used as the α-substituent. These substituents can recognize T-A and A-T base pair of DNA. The invention further provides a new method for producing these preferable substituents. A polyamide integrated with the substituent can strongly recognize a specific gene and delicately control the expression compared with conventional polyamide. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a novel hairpin pyrrole-imidazole polyamide that can recognize a specific gene and is useful for gene therapy and gene diagnosis.

  A pyrrole-imidazole-polyamide composed of two or three different heteroaromatic rings is one of promising synthetic reagents for chemotherapy and biosensing, as well as antibiotics. This compound can recognize a certain DNA sequence and can be introduced into a living cell to control gene expression (Non-patent Document 1). This compound has a hairpin structure, whereby each polyamide ring is connected to each base of DNA and forms a pair adjacent to the DNA string in antiparallel (Patent Documents 1-3). N-methylpyrrole (Py) binds to T, A, and C bases except G base, N-methylimidazole (Im) selects G base, and 3-hydroxyl-N-methylpyrrole (Hp) selects thymine base Is. In this way, if different combinations of three kinds of aromatic amino acids are used, all Watson-Crick base pairs can be recognized (Non-patent Document 2). That is, the Im / Py pair reads G · C base pairs due to symmetry, the Py / Im pair reads C · G base pairs, and the Hp / Py pair is derived from A · T, G · C, C · G base pairs. This is because the T · A base pair can be identified, and the Py / Py pair can distinguish the A · T, T · A base pair from the G · C, C · G base pair.

  However, Hp is easy to decompose, changes with time, and its T base recognition ability is weak compared to Py, so there is a disadvantage in using Hp as a T base recognition element.

  Polyamides composed of aromatic amino acid residues are bent more than the bending of the DNA helix. Due to this difference in curvature, if the number of residues of the polyamide is 5 or less, the polyamide almost matches the pitch of the B-DNA helix and can bind to the minor groove of B-DNA. However, if the polyamide is larger than this, bonding becomes difficult. At this time, by adding β-alanine (β) which is more flexible in the polyamide chain, the orientation structure of the polyamide can be more matched to the curvature of B-DNA. Moreover, the β / β pair has the feature that it can recognize T · A and A · T base pairs non-selectively, similarly to the Py / Py pair (Non-patent Document 3).

  It is disclosed that such a useful polyamide can be produced from monomer units by a solid phase synthesis method (Patent Document 4).

JP 2003-261541 A WO00 / 15642 Publication Special Table 2001-513759 WO2003 / 000683 Publication T. P. Best and 3 others, “Nuclear localization of pyrrole-imidazole polyamide-fluorescein conjugates in cell culture”, Proceedings of the National Academy of Science, USA, 2003, 100, 12063-12068. S. White and four others, “Recognition of the four Watson-Crick base pairs in the DNA minor groove by synthetic ligands”, Nature, 1998, 391, 468-471. J. W. Trauger and three others, "Extension of sequence-specific recognition in the minor groove of DNA by pyrrole-imidazole polyamides to 9-13 base pairs", Journal of the American Chemical Society, 1996, 118, 6160-6166.

  In order to develop a useful reagent having a high DNA recognition ability, it is necessary to search for a substituent having a base recognition ability and a flexibility to match the orientation structure of polyamide with the curvature of B-DNA. Various 5-membered heteroaromatic rings have been investigated, but no suitable one has been found so far.

In order to solve this problem, it has been discovered that an α-substituted ω-aminoalkylcarboxylic acid group that specifically recognizes a T base may be introduced into a polyamide structure. Here, the ω position represents a terminal position opposite to the α position.
Among α-substituted ω-aminoalkylcarboxylic acids, (R) -α, γ-diaminobutyric acid (hereinafter abbreviated as γRN), (S) -α, γ-diaminobutyric acid (hereinafter abbreviated as γSN), (R)- α-hydroxyl-γ-aminobutyric acid (hereinafter abbreviated as γRO) and (S) -α-hydroxyl-γ-aminobutyric acid (hereinafter abbreviated as γSO) can be particularly preferably used. These can be easily incorporated into the polyamide chain and can recognize the T base.

Among these, the α-hydroxyl-ω-aminoalkylcarboxylic acid having S configuration or R configuration is specifically represented by the general formula (1).
(In the formula, C ^* is an optically active carbon atom, which takes S configuration or R configuration, n represents a natural number, and Fmoc represents a fluorenylmethoxycarbonyl group. A hydrogen atom bonded to a carbon atom of the main chain. (The hydrogen atom in the molecule may be replaced with a deuterium atom.)
Of the alkyl carboxylic acids represented by the general formula (1), those with n = 1 are called butyric acid. In this specification, the name butyric acid is used when n = 1 is specified.

The optically active 2-t-butyloxy- (3 + n)-(9-fluorenylmethoxy) carbonylaminocarboxylic acid having this S configuration or R configuration was newly synthesized by the following method.
Starting from optically active (3 + n) -amino-2-hydroxyalkylcarboxylic acid,
(a) The starting material is reacted with (9-fluorenylmethoxy) carbonyl succinimide in a mixture of water and 1,2-dimethoxyethane in the presence of a small amount of sodium bicarbonate to give optically active (3 + n)- [(9-Fluorenylmethoxy) carbonylamino] -2-hydroxyalkylcarboxylic acid was obtained,
(b) Suspend the alkyl carboxylic acid in dichloromethane, add excess isobutene at room temperature and continue the reaction until a clear solution is obtained, 2-t-butoxy- (3 + n)-(9-flux (Oleenylmethoxy) carbonylaminoalkylcarboxylic acid t-butyl ester
(c) A highly active silyl substitution reagent, trimethylsilyl trifluoromethanesulfonic acid (TMSOTf), is added to the butyl ester and reacted.
This gave the desired optically active 2-t-butyloxy- (3 + n)-(9-fluorenylmethoxy) carbonylaminoalkylcarboxylic acid.

  Optically active 2-t-butyloxy-4- (9-fluorenylmethoxy) carbonylaminobutyric acid can be similarly synthesized using optically active 2-hydroxy-4-aminobutyric acid as a starting material.

By using these aminoalkyl carboxylic acids, a hairpin type polyamide represented by the general formula (2) containing a substituent that recognizes the T base in the polyamide structure can be obtained.
(In the formula, X and Y are substituents selected from the group consisting of β-alanine, diaminoalkylcarboxylic acid, optically active α, ωdiaminoalkylcarboxylic acid, and optically active αhydroxyl-ωaminoalkylcarboxylic acid. W is a substituent selected from (CH ₂ ) _r , β alanine, diaminoalkyl carboxylic acid, optically active α, ω aminoalkyl carboxylic acid, optically active α hydroxyl-ω aminoalkyl carboxylic acid. , P, q, r, s, and t are each independently a natural number, and Z represents a terminal diamino group or an alkylation reaction site.)
In general formula (2), all or part of the pyrrole ring may be replaced with an imidazole ring.

  If an alkylation reaction site is used as Z, expression of the DNA can be controlled by alkylating a specific position of the DNA. Examples of such alkylation reaction sites include the DU-86 residue described as [Chem. 11] in Patent Document 1, and the 1,2, 9,9a-tetrahydrocyclopropa [e] benz [e] indol-4-one residue.

In the above compound (2), r = 3, p = 2, q = 1, s = 2, t = 1, including three pyrrole rings and three imidazole rings, and W is (CH ₂ ) ₃ A hairpin type polyamide represented by the following structural formula (3) in which Z is a propyldiamine cation group can be particularly preferably used.
(In the formula, X and Y are substituents selected from β-alanine, diaminobutyric acid, optically active α, γdiaminobutyric acid, and optically active αhydroxyl-γaminobutyric acid, which are equal to or different from each other.)

In the above compound (2), r = 3, p = 2, q = 1, s = 2, t = 1, including three pyrrole rings and three imidazole rings, and W is optically active α, γ A hairpin type polyamide represented by the following structural formula (4), which is a substituent selected from diaminobutyric acid and optically active α-hydroxyl-γ-aminobutyric acid, and Z is a propyldiamine cation group, is particularly preferably used. it can.
(In the formula, X and Y are substituents selected from β-alanine, diaminobutyric acid, optically active α, γdiaminobutyric acid, and optically active αhydroxyl-γaminobutyric acid, which are equal to or different from each other.)

  These polyamides can recognize DNA effectively and stably. In particular, the A-T base pair or the T-A base pair can be recognized.

  The above-mentioned method for synthesizing optically active 2-t-butyloxy-4- (9-fluorenylmethoxy) carbonylaminobutyric acid has an optical property of starting material optically active 2-hydroxy-4-aminobutyric acid with an accuracy of 97% or more. It has the characteristic that it is not changed by. Moreover, the yield is close to 70%, which is extremely excellent.

  This α, γ-substituted aminobutyric acid residue can be easily incorporated between the pyrrole-imidazole aromatic ring, can specifically recognize the T base, and has flexibility, so that polyamide can be used as a hairpin. It can be combined more flexibly with type DNA. Therefore, the polyamide containing this residue can recognize a specific gene more strongly and control its expression more skillfully than known polyamides.

(Abbreviation)
In the following description, compound names are indicated by abbreviations for simplicity of description. The abbreviations 1-12 represent the 12 polyamides shown in FIG. 5, and the abbreviations 25-28 represent the 4 polyamides shown in FIG. The abbreviation 13-17 represents each compound shown in FIG. 1, and the abbreviation 19-24 represents each compound shown in FIG.

(Monomer synthesis)
Optically active monomers γSO17a (S) and γRO17b (R) with protecting groups were synthesized by the following method. FIG. 1 shows the chemical reaction formula.
The raw material (2S) -2-hydroxy-4- (9-fluorenylmethoxy) carbonylaminobutyric acid 14a was prepared in the presence of a small amount of sodium bicarbonate in a mixture of water and 1,2-dimethoxyethane. Synthesized from the corresponding optically active starting material 13a by reacting with (olenylmethoxy) carbonyl succinimide. The produced Fmoc-protected 2-hydroxybutyric acid 14a was suspended in DCM (dichloromethane) and excess isobutene was added at room temperature. The reaction was continued until a clear solution was obtained to give (2s) -2-butoxy-4- (9-fluorenylmethoxy) carbonylaminobutyric acid 15a. To cleave t-butyl ester from 15a, a highly active silyl substitution reagent TMSOTf (trimethylsilyl trifluoromethane sulfonic acid) is used to cross (2S) -2-tbutoxy-4 -(9-Fluorenylmethoxy) carbonylaminobutyric acid 17a was obtained. 17b could be synthesized in the same way. The optical isomer purity of these compounds measured by NMR was 97% or more for both 17a and 17b.

In compounds 17a and 17b, the base-labile amino protecting group Fmoc and the acid-labile hydroxyl protecting group t-butyl coexist, making it difficult to obtain optimal synthesis conditions for optically active 17a and 17b. It was. There are two important steps to avoid side reactions and racemization and to obtain optically active products. One is the simultaneous formation of t-butyl ester and ether, and the second is to convert t-butyl ester to the corresponding trimethylsilyl ester. This synthesis was made possible by the successful selective protection of hydroxyl groups by t-butyl and the selective deprotonation of t-butyl esters by optically active hydroxyamino acids.
This new synthesis method was not known at all, and thereby the racemization was suppressed to 3% or less, and the final product 17a or 17b could be obtained in a yield of 69.5% or 67.2%.

  This monomer production method is not limited to aminobutyric acid and can be generally applied to aminocarboxylic acids.

(Polyamide synthesis)
In order to investigate the T base recognition ability of α-substituted γ-butyric acid, using Fmoc-β-alanine clear acid resin and 8 types of monomers (17a, 17b, 19-24) shown in FIG. Sixteen polyamides were synthesized by the Fmoc solid phase synthesis method. DMF (N, N-dimethylformamide) as solvent, HATU (N- (dimethylamino-1H-1,2,3triazo [4,5-b] -pyridino-1-ylmethylene)- N-methylmethanaminium-6-fluorophosphoric acid) was used.

  Fmoc-Py acid 19 is activated by HATU in the presence of DIEA (N, N-diisopropylethylamine), and is easily combined with swollen resin 18 in the synthesis column when reacted for about 60 minutes. Aromatic monomer 20 can also be coupled in this manner. Modified or unmodified aliphatic monomers 17a, 17b, 21, 22, 23, and 24 can be similarly bonded using HATU. Regarding aliphatic monomers 17a, 17b, 23, and 24, there was no difference depending on whether they were t-butoxy group or t-butoxycarbonylamino group (NHBoc). In order to obtain polyamide in a high yield, a double bond cycle was essential for the bond between the monomer and imidazole and the bond between the imidazole itself.

  After the solid phase synthesis by the synthesis machine is completed, the polyamide is separated from the resin 1a-12a by aminolysis with Dp (3-dimethylaminopropylamine) and the desired polyamide 1,2,3,4, t-butyl protected polyamide 5 , 6,7,8, Boc-protected polyamide 9,10,11,12 (Boc: t-butoxycarbonyl). Racemization did not occur during the elimination of t-butoxy by cleavage of the bond between oxygen and t-butyl. It was confirmed that the final purity of all synthetic polyamides was 98% or higher by combining the results of HPLC, H-NMR and mass spectrometry.

A polyamide in which W is any one of (CH ₂ ) _r , β alanine, diaminoalkyl carboxylic acid, optically active α, ω aminoalkyl carboxylic acid, optically active αhydroxyl-ω aminoalkyl carboxylic acid in the general formula (2), It can be synthesized in the same manner as above and the purity can be confirmed in the same manner.

(Surface plasmon resonance test)
34-base oligonucleic acid (5'-biotin-GGCCGATG N 'GCATGCTATAGCATGC N CATCGGCC-3': N · N) noncovalently bonded to a Biacore sensor chip functionalized with streptavidin 'Represents any one of T · A, A · T, C · G, and G · C.) And the dynamic and thermodynamic properties of the binding of polyamide to DNA The surface plasmon resonance method was used from the viewpoint of steric properties. The results are shown in FIG. As shown in FIG. 3 (a) or (b), this oligonucleic acid has a binding site with a polyamide having a T-base selective recognition site, and forms a hairpin type DNA fixed at two points.

  In FIG. 3, X / Y is an aliphatic monomer pair, and β / β, β / γ, γ / β, γ / γ, β / γSO, β / γRO, γSO / β, γRO / β, β / It represents either γSN, β / γRN, γSN / β, or γRN / β. N · N ′ represents one of T · A, A · T, C · G, and G · C. ● represents Im, ○ represents Py, β represents a β-alanine residue, Dp represents 3-dimethylaminopropylamide, ⊂ represents a γ-aminobutyric acid residue connecting two subunits, and Ac represents acetyl.

In FIG. 4, each experiment was repeated three times to fit a 1: 1 interaction model. All experiments were performed with the same amount of DNA on the sensor chip using HBS-EP buffer solution.
A: Pair of polyamide 1 having β / β pair and A · T base pair. Concentrations range from 3.75 x 10 ^-8 M (lowest curve) to 2.00 x 10 ^-7 M (highest curve). B: Pair of polyamide 5 having β / ^S γ _α OH pair and A · T base pair. Concentrations range from 2.00 x 10 ^-8 M (lowest curve) to 5.00 x 10 ^-7 M (highest curve). C: A pair of polyamide 5 having a β / ^S γ _α OH pair and a T / A base pair. Concentrations range from 1.00 × 10 ⁻⁸ M (lowest curve) to 1.00 × 10 ⁻⁷ M (highest curve). D: Pair of polyamide 9 having β / ^S γ _α NH2 pair and A · T base pair. Concentrations range from 1.00 × 10 ⁻⁸ M (lowest curve) to 5.00 × 10 ⁻⁷ M (highest curve). E: Pair of polyamide 9 having β / ^S γ _α NH2 pair and T / A base pair. Concentrations range from 1.50 x 10 ^-7 M (lowest curve) to 1.25 x 10 ^-6 M (highest curve).

The surface plasmon resonance of polyamide 1-12 was measured with 0.1% HBS-EP buffer (0.01M HEPES, pH 7.4, 0.15M NaCl, 3mM EDTA, 0.005% Surfactant P20) using Biacore X instrument and SA sensor chip. In a solution with DMSO (dimethyl sulfoxide) added at 25 ° C. As shown in Fig. 4, the dissociation equilibrium constant (K _D , M) is calculated from functionalized γ monomer / β and T · A, A · T, G · C, C · G. It was determined by fitting to a theoretical model that reveals the affinity and specificity of binding to four base pairs. Standard free energy changes from ^{_{K D (△ G 0 kcal /}} mol) was determined also.

In order to obtain reasonable and accurate binding constants, surface plasmon resonance measurements were performed at various concentrations for each polyamino. The quantitative relationship of the polyamide binding to the hairpin DNA was determined by comparing the maximum response RUmax when one molecule of polyamide was bound to one binding locus with the response RU at saturation. In the single seat model
RUmax = (RU _DNA / MW _DNA ) × MW _polyamide × RII
It becomes. Here, RU _DNA is the amount of hairpin DNA immobilized on the sensor chip, MW is the molecular weight of polyamide and hairpin DNA, and RII is the ratio of the refractive index increase of the compound to the refractive index of DNA. RII takes a value from 1.25 to 1.40.

(Recognition of stereochemistry of hydroxyl group and effect of bond on stereochemistry)
To assess the cognition of polyamides 1-12 and 25-28 containing aliphatic monomers that pair with T • A, A • T, G • C, C • G base pairs in the DNA minor groove The characteristics were investigated by surface plasmon resonance method. The pair creation model is shown in FIG. 3, and the results are shown in FIGS.
5 and 6, the experimental error is less than 10%. Selectivity was defined as K _D (T · A) / K _D (A · T).
In the following DNA base sequence formula, the underlined portion indicates the binding position of the polyamide.

It should be noted that in all the polyamides examined, 5′-TGC N CA-3 ′ (N = C, G) does not bind at low concentrations, but has high non-selective binding at high concentrations. Let's go. As a result, an accurate dissociation equilibrium constant could not be obtained. For polyamides 1-4 with unsubstituted aliphatic amino acid pairs, 5'-TGC N CA-3 '/ 3'-ACG N ' GT-5 '(N ・ N' = T ・ A and A・ Selectivity with T) was not observed.
However, when γ-aminobutyric acid (hereinafter abbreviated as γ, the same as in FIG. 3 (a)) is introduced as in polyamides 2 and 3 to form a pair with β-alanine (hereinafter abbreviated as β), Note that it was found that binding to AT base pairs was reduced 35-fold and 4.7-fold and binding to TA base pairs was reduced 30-fold and 4.5-fold compared to the β / β pair. It is.
Furthermore, in polyamide 4 in which two βs are substituted with two γs, the binding property to 5′-TGC A CA-3 ′ / 3′-ACG T GT-5 ′ is 846 times greater, and 5′-TGC T CA- Binding with 3 '/ 3'-ACG A GT-5' is reduced by 724 times. From these measurement results, it can be seen that when one γ is inserted instead of β to recognize an AT base pair and a TA base pair, the binding affinity is decreased, but not a large decrease.

As shown in FIG. 5, polyamide 5 having β / γSO is 5′-TGC from 5′-TGC T CA-3 ′ / 3′-ACG A GT-5 ′ (N · N ′ = T · A). A CA-3 '/ 3'-ACG T GT-5' (N · N '= A · T) is 19 times greater. Comparison of the binding affinity of β / γSO and γSO / β in polyamides with aliphatic monomers, such as polyamides 5 and 7, for polyamide 5-12 reveals the following. α-Hydroxy- / amino substituted γ monomers cannot recognize T and A bases due to the hydrogen bond between OH / NH ₂ and O ₂ of T, but perhaps two bases at the same time, namely 5'-TG- 3 ′ is recognized through a strong hydrogen bond between OH / NH ₂ and N3 of the 3′-G base adjacent to the T base (FIG. 5).

Furthermore, the fact that the hydroxy substituent of the γ monomer is in the α position predicts that there is another hydrogen bond between N3 of the 3 ′ terminal guanine adjacent to the T base and this OH group. This is the reason for the bond selectivity between polyamide 5 and 5'-TGC N CA-3 '/ 3'-ACG N ' GT-5 '(N ・ N' = T ・ A and A ・ T). Wax (Figure 7). In addition, van der Waals force acts between the proton at the α position of the γ residue and H1 ′ and / or H4 ′ of the deoxyrepose of guanine. Taken together, this implies that binding affinity and selectivity are directly related to the neighboring base. In this case, when polyamide 5 having one β / γSO pair and 5′-TGC T CA-3 ′ / 3′-ACG A GT-5 ′ are combined, a large steric hindrance by the A base is caused by the 3 ′ terminal. It weakens the additional hydrogen bond between N3 and hydroxyl group of guanine, leading to weaker affinity. Therefore, in recognition, the monomer γSO actually distinguishes one 5′-TG-3 ′.

FIG. 7 shows a molecular model of the interaction between polyamide 5 having a β / γSO pair and DNA (5′-GCATGC A / T CATCG-3 ′ / 5′-CGATG A / T GCATGC-3 ′).
(a) Molecular model of 1: 1 complex of polyamide 5 and 5′-GCATGC A CATCG-3 ′ / 5′-CGATG T GCATGC-3 ′ sequence. Obtained by calculation of the energy minimum method. Polyamide is shown in bold lines.
(b) Partial enlarged view of a molecular model of polyamide 5 and A · T base pairs. (c) The figure which showed the hydrogen bond in (b). (d) Partial enlarged view of a molecular model of polyamide 5 and T / A base pairs. (e) The figure which showed the hydrogen bond in (d).

In order to clarify the difference in bonding between polyamides quantitatively, the standard free energy change (ΔG ⁰ ), which is a thermodynamic parameter for complex formation, was determined from the results of surface plasmon resonance (FIG. 5). The binding between 5 and a sequence having A · T base pairs is more stable by about 2 kcal / mol than the binding with a sequence having T · A base pairs. The more stable binding with the 5'-TGC A CA-3 '/ 3'-ACG T GT-5' sequence is the reason why the 5'-TGC T CA-3 '/ 3'-ACG A GT-5' sequence is more stable. This is associated with a slower dissociation rate compared to binding (FIGS. 4B, 4C). The dissociation rate of the complex of polyamide 5 and 5'-TGC A CA-3 '/ 3'-ACG T GT-5' is slower because the hydrogen bond between the OH group and N of G is strong. Based on difficulties, it may be related to the release and uptake of water molecules in the DNA minor groove.

On the other hand, for polyamide 6 with γRO monomer, 5'-TGC A CA-3 '/ 3'-ACG T GT-5' and 5'-TGC T CA-3 '/ 3'-ACG A GT-5' When comparing recognition, the selectivity increased only 4.8-fold (Figure 5 and Figures 4A and B). In 6 in which γSO of 5 was replaced with γRO at the same position, 5′-TGC A CA-3 ′ / 3′-ACG T GT-5 ′ (N ・ N = T ・ A) and 5′-TGC T CA- The bond to 3 '/ 3'-ACG A GT-5' became unstable. In particular, when γRO was opposed to T, the affinity decreased 4.3 times. This is because the (R) α hydroxyl group faces the wall direction of the minor groove of B-DNA and sterically collides with the wall, while the (S) α hydroxyl group has a more favorable steric orientation. Therefore, it can be explained that the binding affinity increases.

Conversely, polyamides 7 and 8 have γSO / β and γRO / β pairs, respectively, 5′-TGC A CA-3 ′ / 3′-ACG T GT-5 ′ and 5′-TGC T CA-3 ′. / 3'-ACG A GT-5 'binds with little selectivity. However, the binding affinity is greater compared to polyamide 5 with the corresponding β / γSO pair and polyamide 6 with the β / γRO pair. This implies that the van der Waals force between the γ monomer having a hydroxyl group and the base on the side makes an important contribution to the selectivity.

Furthermore, because polyamide 5-12 with substituted γ groups is unstable, 5'-TGC A CA-3 '/ 3'-ACG T GT-5' compared to polyamide 1 with β / β pair And 5'-TGC T CA-3 '/ 3'-ACG A GT-5' sequence more clearly recognized. However, compared to polyamide 1, some polyamides are destabilized while others are not (FIG. 5). This seems likely to be explained as the selectivity in the sequence selective recognition of polyamide DNA increases at the expense of binding affinity.

  The above results are all compatible with the 1: 1 interaction model of polyamide and DNA, except for the recognition of G · C base pairs and C · G base pairs. Despite the use of high concentrations of polyamide, no example of 2: 1 binding between polyamide and hairpin DNA could be confirmed.

  It is shown in FIG. 6 that polyamide 25-28 having γRO, γSO, γRN, and γSN in the hairpin portion can recognize DNA by binding to the minor groove of DNA in the same manner as polyamide 1-12.

(Effects on binding of amino substituents)
Polyamide 9 with β / γSN pair is 5'-TGC A CA-3 '/ 3'-ACG T GT-5' sequence from 5'-TGC T CA-3 '/ 3'-ACG A GT-5' To 9.6 times stronger. In contrast, 10 with a β / γRN pair binds more than 2.3 times to the 5′-TGC A CA-3 ′ / 3′-ACG T GT-5 ′ sequence (FIG. 5). This difference in selectivity can be explained by the fact that the difference in the orientation of the bond between the S amino group and the R amino group results in a difference in recognition ability. Furthermore, from the results of surface plasmon resonance shown in Fig. 4D and Fig. 4E, the rapid dissociation of 9 and the T • A sequence complex indicates that the binding affinity between sequences with A • T and T • A base pairs. It can be attributed to gender differences. Similarly, as shown in FIG. 5, the ΔG value corresponds to that polyamide 9 is more likely to bind to A · T base pairs than to T · A base pairs.

Similarly, the binding affinities between 11 with γSN / β and 12 with γRN / β and A · T and T · A base pairs were also found to be nearly equal. However, its binding stability was greater than 9 with γSN / β and 10 with γRN / β. Furthermore, polyamide 5 with β / γSO is 5'-TGC A CA-3 '/ 3'-ACG T GT-5' or 5'-TGC T CA-3 '/ 3' than 9 with β / γSN. -ACG A GT-5 'has a higher binding affinity, particularly when there is a γSO monomer on the T base side, showing an increase of about 4.6 times. The difference is that (S) α-hydroxy forms a stronger hydrogen bond with N3 of guanine than protonated (S) α-amino, and large protonated amino collides sterically with minor grooves, or DNA This can be explained as causing a large distortion in the minor groove. In short, hydrogen bonding ability is an important factor, but DNA minor grooves, surface complexity of hairpin polyamides and van der Waals interactions also play a role in polyamide minor groove recognition.

(Molecular model)
The mechanism by which polyamide 5 binds selectively and stronger to A / T base pairs than T / A base pairs is clear when molecular models of 5-DNA complexes are determined by the energy minimum method. The results are shown in FIG. As shown in Figure 7a, hairpin polyimide 5 is antiparallel to the 5'-GCATGC A CATGC-3 '/ 5'-CGATG T GCATGC-3' sequence with A / T base pairs in the DNA minor groove. It has been shown that it fits well and is in close contact with the base to be recognized. 7b and 7d show that the α-hydroxy of the γ monomer is close to N3 of 3'-G, and the γ residue and G base are closely matched to each other. Shows that hydrogen bonds are formed. When 5 recognizes A · T and T · A base pairs, the length of this hydrogen bond was 2.53 and 4.11 respectively (see FIGS. 7C and 7D).

  However, as shown in Figure 7d, in the case of the 5-T complex, a large three-dimensional structure of the A base is seen in the 5-A complex, pushing the γ monomer from the bottom of the minor groove (Fig. 7b) prevents the formation of strong hydrogen bonds and affects the preferred aromatic ring overlap between the polyamide monomer and the DNA base. As a result of these effects, the binding affinity between 5 and the T • A complex is weaker. In conclusion, the molecular model is in good agreement with the surface plasmon resonance results, and the results obtained thermodynamically are based on the molecular level.

In conclusion, different aliphatic monomer pairs (β / β, β / γ, γ / β, γ / γ, β / γSO, β / γRO, γSO / β, γRO / β, β / γSN, β / γRN, γSN Twelve new polyamides with / β, γRN / β) were designed and synthesized, and the kinetic properties of their binding to hairpin DNA were investigated by surface plasmon resonance. Importantly, the three-dimensional structure of the α-substituent of the γ-aminobutyric acid group of the polyamide has been found to affect the binding to the DNA sequence compared to unmodified β-alanine or γ-aminobutyric acid. More importantly, polyamides with β / γSO pairs are 5'-TGC A CA-3 '/ 3'-ACG T than 5'-TGC T CA-3' / 3'-ACG A GT-5 '. Polyamides with β / γSN bind to TG-5 'with a selectivity of 19 times greater, while polyamides with β / γSN bind to these sequences with a selectivity of 9.6 times.

  The recognition by hairpin polyamides with α-substituted γ-aminobutyric acid in the DNA minor groove is in the molecular environment such as the base on the side of the substituent at the α-position of γ-aminobutyric acid, the three-dimensional structure, the three-dimensional size, the electronic and hydrogen bonding ability. It is greatly affected. Combined with surface plasmon resonance and molecular model results, α-hydroxy modified γ-aminobutyric acid is not only a flexible subunit of polyamide structure, but also recognizes 5'-TG-3 'in hairpin polyamides Indicates a unit.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited at all by these Examples.

(General experimental conditions)
¹ H NMR spectra were measured at 400 MHz with a JEOL JNM-FX400 superconducting NMR spectrometer. At this time, the chemical shift is expressed in ppm based on the solvent, and the coupling constant is expressed in hertz. In the NMR results shown below, s is a single line, d is a double line, t is a triple line, q is a four line (qluartet), and m is a multiplet. ), Br indicates a broad line. Moreover, the underline drawn by H shows that it can belong to the signal of the hydrogen atom among several hydrogen atoms.

The UV-visible absorption spectrum was measured using a buffer solution with a Beckman Coulter DU650 type diode array spectrophotometer.
MALDI-TOF spectra were measured with a Bruker Daltonics BioTOF II mass spectrometer and ESI-mass spectra were measured with an Applied Biosystems API 165 mass spectrometer.

Thin layer chromatography (TLC) was performed on a glass plate covered with 60F254 (Merck) silica gel, and column chromatography was performed using silica gel 60 (63-200 μm). Visualization was by ultraviolet light irradiation or ninhydrin spray.
For high performance liquid chromatograph (HPLC), Tosoh Corporation CCP & 8020 series system was used. For the analysis, a 150 x 10 mm Chemco Pak Chemocoband 5-ODS-H reverse phase column was used, a solvent mixture of water and acetonitrile containing 0.1% acetic acid was used, and the flow rate was 3.0 mL per minute, at 254 nm. Detected. For synthesis, a 150 x 20 mm YMC-Pack R & D ODS DOD-5-ST S-5 reverse phase column was used, and a solvent mixture of water and acetonitrile containing 0.1% acetic acid was used, and the flow rate was 1 min. 6.0 mL and detected at 254 nm or 350 nm. The fractionated components were analyzed by ESI or TOF mass analyzer.

  The Biacore test was performed using the Biacore X system, and data analysis was performed using BIA evaluation version 4.1. As pure water, 18 MΩ pure water was obtained from Millipore MilliQ pure water production equipment. The Biacore test water was further filtered at 0.3 μm and degassed before use.

  Hairpin DNA was obtained from Proligo and used without purification. HBS-EP buffer and sensor chip were purchased from Biacore AB (Sweden). Clear resin, optically active (S) and (R) -α-t-butoxycarbonylamino-γ- (9-fluorenylmethoxy) -carbonylaminobutyric acid were purchased from Peptides International. Fmoc-ImCOOH, Fmoc-PyCOOH, DMF, DMSO, piperidine and pyridine were purchased from Wako, and Fmoc-β-Ala-OH and Fmoc-γ-Abu-COOH were purchased from Novabiochem. Fmoc is an abbreviation for fluorenylmethoxycarbonyl group. (S) -α-hydroxyl-γ-aminobutyric acid (γSO) was purchased from Aldrich and (R) -α-hydroxyl-aminobutyric acid (γRO) was purchased from Xiamen Forever Green Source Bio-Chem Technology. Other chemicals were purchased from standard reagent suppliers.

(Monomer synthesis: (2S) -4- (9-fluorenylmethoxy) carbonylamino-2-hydroxybutyric acid 14a and its (2R) form 14b)
(9-Fluorenylmethoxy) carbonyl succinimide (Fmoc-ONSu) (6.72 g, 24 mmol) was dissolved in 90 mL of 1,2 dimethoxyethane and (S) -4-amino-2-hydroxybutyric acid 13a (2.38 g 24 mmol) and sodium bicarbonate (2.0 g) were added dropwise to an aqueous solution of 30 mL of water with stirring at room temperature over 45 minutes. The solution was then kept stirring for 15 hours. The progress of this reaction was monitored by thin layer chromatography using DCM / methanol / acetic acid (4/1 / 0.2 v / v) solvent. The solution was acidified with concentrated hydrochloric acid and then concentrated on a rotary evaporator.
20 mL of ice water was added to the residue, filtered, and washed with a small amount of cold water to obtain a white solid. The filtrate was similarly concentrated, filtered by adding cold water, and a white solid was obtained in the same manner. These were combined and dehydrated to give a white solid in 98.2% yield. The purity was confirmed to be 98% by HPLC. It was confirmed by ESI mass spectrometry that this white solid was 14a.

The properties of 14a are as follows.
Rf: 0.70 (dichloromethane / methanol / acetic acid, 4/1 / 0.2 v / v).
HPLC: t _R = 19.73min.
¹ H NMR (400 MHz, DMSO-d ₆ ): δ 7.87 (d, J = 7.6 Hz, 2H, Fluoren H), 7.67 (d, J = 7.2 Hz, 2H, Fluoren H), 7.40 (m, 2H, Fluoren H), 7.32 (m, 2H, Fluoren H), 4.25 (m, 2H, OCH ₂ ), 4.22 (m, 1H, Fluoren H), 3.97 (dd, J = 8.8Hz, J = 4.0Hz, 1H, CH ₂ C H ), 3.10 (m, 2H, NHC H ₂ ), 1.78 and 1.59 (m, 2H, C H ₂ CH).
ESI-MASS: calculated for C ₁₉ H ₁₉ NO ₅ 341.13, found 342.12 [M + H] ⁺ .

Compound 14b was synthesized in the same manner as Compound 14a. The yield was 95.8% and the purity judged by HPLC was 98%. The properties of 14b are as follows.
Rf: 0.71 (dichloromethane / methanol / acetic acid, 4/1 / 0.2 v / v).
HPLC: t _R = 19.75min.
¹ H NMR (400 MHz, DMSO-d ₆ ): δ 7.87 (d, J = 7.6 Hz, 2H, Fluoren H), 7.67 (d, J = 7.2 Hz, 2H, Fluoren H), 7.40 (m, 2H, Fluoren H), 7.32 (m, 2H, Fluoren H), 4.26 (m, 2H, OCH ₂ ), 4.20 (m, 1H, Fluoren H), 3.96 (dd, J = 8.7Hz, J = 4.0Hz, 1H, CH ₂ C H ), 3.08 (m, 2H, NHC H ₂ ), 1.79 and 1.59 (m, 2H, C H ₂ CH).
ESI-MASS: calculated for C ₁₉ H ₁₉ NO ₅ 341.13, found 342.12 [M + H] ⁺ _, 364.12 [M + Na] ⁺ .

((2S) -2-t-butoxy-4- (9-fluorenylmethoxy) carbonylaminobutyric acid t-butyl ester (15a) and its (2R) form (15b))
Using a pressure flask, 14a (1 g, 2.93 mmol) was dissolved in 20 mL of dry dichloromethane (DCM) containing 40 μL of concentrated sulfuric acid (98%), and 20 mL of isobutylene was added to the solution while stirring at low temperature. The solution was kept stirring for 40 hours while gradually warming to room temperature. The progress of the reaction was monitored by TLC using an ethylacetone-hexane mixed solvent (1/2, v / v). After completion of the reaction, the solution was exposed to air at room temperature to remove excess isobutylene. The solution was then transferred to a separatory funnel, washed with 3 mL of water and brought to pH 7.0 with 2M sodium hydroxide. The dichloromethane layer was separated and the organic solvent was carefully removed by rotary evaporation. Ethyl acetate (5 mL) and water (3 mL) were added to the residue, and the organic layer was washed with Brine (3 mL), dried over anhydrous sodium sulfate for 5 hours, filtered, dried until free of solvent, 1.41 g of a pale yellow oily crude product was obtained. This product was purified using column chromatography (63-200 μm silica gel) by gradient elution with a mixed solvent of ethyl acetate and hexane to obtain 1.01 g of a pale yellow oil in a yield of 76.5%. The purity as seen by HPLC was 98%. ESI mass spectrometry confirmed that it was the desired compound 15a.

The properties of 15a are as follows.
Rf: 0.52 (ethyl acetate / hexane, 1/2 v / v).
HPLC: t _R = 29.70min.
¹ H NMR (400 MHz, DMSO-d ₆ ): δ 7.88 (d, J = 7.2 Hz, 2H, Fluoren H), 7.67 (d, J = 7.6 Hz, 2H, Fluoren H), 7.40 (m, 2H, Fluoren H), 7.31 (m, 2H, Fluoren H), 7.24 (t, J = 4.8Hz, 1H, NH), 4.28 (m, 2H, OCH 2), 4.20 (m, 1H, Fluoren H), 3.88 ( dd, J = 8.8Hz, J = 3.6Hz, 1H, CH ₂ C H ), 3.04 (m, 2H, NHC H ₂ ), 1.67 and 1.58 (m, 2H, C H ₂ CH), 1.39 (s, 9H , COOCMe ₃ ), 1.09 (s, 9H, OCMe ₃ ).
ESI-MASS: calculated for C ₂₇ H ₃₅ NO ₅ 453.25, found 454.26 [M + H] ⁺ .

15b was synthesized in the same manner as described above, and the yield was 79.6% and the purity observed by HPLC was 98%. The properties of 15b are as follows.
Rf: 0.53 (ethyl acetate / hexane, 1/2 v / v).
HPLC: t _R = 29.70min.
¹ H NMR (400 MHz, DMSO-d ₆ ): δ 7.88 (d, J = 7.6 Hz, 2H, Fluoren H), 7.66 (d, J = 7.6 Hz, 2H, Fluoren H), 7.40 (t, 2H, Fluoren H), 7.31 (m, 2H, Fluoren H), 7.23 (t, J = 4.8Hz, 1H, NH), 4.28 (m, 2H, OCH 2), 4.19 (t, J = 6.8Hz, 1H, Fluoren H), 3.89 (dd, J = 8.8 Hz, J = 4.0 Hz, 1H, CH ₂ C H ), 3.03 (m, 2H, NHC H ₂ ), 1.66 and 1.57 (m, 2H, C H ₂ CH), 1.39 (s, 9H, COOCMe ₃ ), 1.08 (s, 9H, OCMe ₃ ).
ESI-MASS: calculated for C ₂₇ H ₃₅ NO ₅ 453.25, found 454.27 [M + H] ⁺ .

((2S) -2-t-Butyloxy-4- (9-fluorenylmethoxy) carbonylaminobutyric acid (17a) and its (2R) form (17b))
15a (295.5 mg, 0.65 mmol) was dissolved in 6.5 mL of dioxane to which 150 μL of N, N′diisopropylethylamine (DIEA) was added, and this solution was stirred and trimethylsilyltrifluoromethanesulfonic acid (TMSOTf, 215.6 mg, 0.97 mmol) Was added dropwise over 25 minutes at room temperature. The solution after the reaction was further stirred for 50 minutes. The progress of the reaction was monitored by TLC using a mixed solvent of ethyl acetate / hexane / acetic acid (2/1 / 0.01, v / v). After completion of the reaction, the reaction solution was cooled to room temperature. Water (5 mL) and ethyl acetate (8 mL) were quickly added to the reaction solution on an ice-water bath, and the solution was transferred to a separatory funnel. The organic layer was separated and washed with 5% aqueous sodium bicarbonate.
The reaction intermediate 16a is extremely unstable and is hydrolyzed during operation to the target compound 17a. Therefore, without separating and purifying the resulting mixed solution, 10% hydrochloric acid was added to adjust the pH to 5, and stirring was continued for another 2 hours. The reaction solution was washed with 5% sodium bicarbonate, 10% potassium sulfate, and 5 mL of bryne, dried over sodium sulfate, filtered and further dried to remove the solvent. Thus, 239.3 mg of white solid was obtained in 92.6% yield, and the purity was 98% as seen by HPLC. The final product was confirmed to be 17a by ESI mass spectrometry.

The properties of 16a are as follows.
Rf: 0.46 (acetate / hexane / acetic acid, 2/1 / 0.01 v / v).
HPLC: t _R = 25.83min.
ESI-MASS: calculated for C ₂₆ H ₃₅ NO ₅ Si 469.23, found 470.21 [M + H] ⁺ _, 492.23 [M + Na] ⁺ .

The properties of 17a are as follows.
Rf: 0.28 (acetate / hexane / acetic acid, 2/1 / 0.01 v / v).
HPLC: t _R = 24.01min.
¹ H NMR (400 MHz, DMSO-d ₆ ): δ 12.3 (br, 1H, COOH), 7.87 (d, J = 7.6Hz, 2H, Fluoren H), 7.67 (d, J = 7.2Hz, 2H, Fluoren H), 7.40 (t, J = 7.6Hz, 2H, Fluoren H), 7.30 (t, J = 7.6Hz, 2H, Fluoren H), 7.25 (t, J = 4.8Hz, 1H, NH), 4.27 (m , J = 6.4Hz, 2H, OCH ₂ ), 4.19 (t, J = 6.8Hz, 1H, Fluoren H), 3.94 (dd, J = 8.4Hz, J = 3.6Hz, 1H, CH ₂ C H ), 3.04 _{(m, 2H, NHC H 2} ), 1.71 and 1.60 (m, 2H, C H 2 CH), 1.13 (s, 9H, OCMe 3).
ESI-MASS: calculated for C ₂₃ H ₂₇ NO ₅ 397.19, found 398.26 [M + H] ⁺ _, 420.17 [M + Na] ⁺ .

17b was synthesized in the same manner as described above, and the yield was 88% and the purity observed by HPLC was 98%.
The properties of 16b are as follows.
Rf: 0.48 (acetate / hexane / acetic acid, 2/1 / 0.01 v / v).
HPLC: t _R = 25.9min.
ESI-MASS: calculated for C ₂₆ H ₃₅ NO ₅ Si 469.23, found 470.22 [M + H] ⁺ _, 492.25 [M + Na] ⁺ .
The properties of 17b are as follows.
Rf: 0.28 (acetate / hexane / acetic acid, 2/1 / 0.01 v / v).
HPLC: t _R = 24.0min.
¹ H NMR (400 MHz, DMSO-d ₆ ): δ 12.3 (br, 1H, COOH), 7.88 (d, J = 7.6Hz, 2H, Fluoren H), 7.67 (d, J = 7.2Hz, 2H, Fluoren H), 7.40 (t, J = 7.4Hz, 2H, Fluoren H), 7.31 (t, J = 7.6Hz, 2H, Fluoren H), 7.26 (t, J = 4.6Hz, 1H, NH), 4.28 (m , 2H, OCH ₂ ), 4.19 (t, J = 6.0Hz, 1H, Fluoren H), 3.95 (dd, J = 8.2Hz, J = 3.6Hz, 1H, CH ₂ C H ), 3.05 (m, 2H, NHC H ₂ ), 1.71 and 1.60 (m, 2H, C H ₂ CH), 1.11 (s, 9H, OCMe ₃ ).
ESI-MASS: calculated for C ₂₃ H ₂₇ NO ₅ 397.19, found 398.23 [M + H] ⁺ _, 420.16 [M + Na] ⁺ .

  The above synthesis scheme is shown in FIG. The reagents at each synthesis stage are as follows. (i) (S)-(-)-4-amino-2-hydroxybutyric acid (13a) or (R)-(+)-4-amino-2-hydroxybutyric acid (13b), sodium bicarbonate , Water, (ii) FmocONSu, 1,2 dimethoxyethane, (iii) concentrated hydrochloric acid, (iv) 98% sulfuric acid, DCM, isobutylene, (v) 2M aqueous sodium hydroxide, (vi) TMSOTf, DIEA, dioxane, ( vii) ethyl acetate aqueous solution, (viii) 10% hydrochloric acid, (ix) 5% sodium hydrogen carbonate and 10% potassium sulfate aqueous solution.

(Determination of enantiomeric purity of 17a and 17b)
17a or 17b (7.95 mg, 0.02 mmol) is dissolved in N, N dimethylformamide (0.45 mL), and N- (dimethylamino-1H-1,2,3 triazo [4,5-b] -pyridino-1- ylmethylene) -N-methylmethanaminium hexafluorophosphate (HATU) (22.8 mg, 0.06 mmol) and N, N-diisopropylethylamine (DIEA) (4.6 μL) were added. The mixture was stirred at room temperature for about 15 minutes, after which (S)-(−)-1- (1-naphthyl) ethylamine (7.78 mg, 0.0454 mmol) was added. The mixture was subsequently stirred at room temperature for 2 hours. The progress of the reaction was monitored by TLC using an ethyl acetate-hexane mixed solvent (1/1, v / v).

After completion of the reaction, 1 mL of ethyl acetate was added to the solution and washed with 10% citric acid (2 × 1 mL) followed by brine (1 × 1 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by chromatography (63-200 μm silica gel column) using a mixed solvent of ethyl acetate / hexane (2/1, v / v). In this way, 10.9 mg of amide of 17a (S) was obtained in 99% yield, and 10.8 mg of amide of 17b (R) was obtained in 99% yield. The product was confirmed by ¹ H NMR and ESI mass spectrometry. The amide of 17a (S) has a triplet (J = 5.4) at δ7.17ppm, the doublet (J = 8.0) at δ6.83ppm, and the amide of 17b (R) has a triplet at δ7.23ppm (J = 5.6), a double line (J = 7.6) was shown at δ6.96 ppm. There was no evidence of any diastereomeric impurities (<3%) in any spectrum.

(Polyamide synthesis by Fmoc chemistry: mechanical procedure in solid phase)
For machine-based polyamide synthesis, a computer-operated Pioneer peptide synthesizer (Applied Biosystems) was used, and Fmoc chemistry was used to operate at a scale of 0.10 mmol (200 mg clear resin, 0.52 meq / g). The reaction position is as follows. Washing position: N, N-dimethylformamide (DMF), non-blocking position: piperidine in DMF 20%, activation position 1: 0.5 M HATU in DMF, activation position 2: 1.0 MDIEA in DMF, auxiliary reagent position 1: methanol, auxiliary reagent position 3: DMF solution of 5% acetic anhydride and 5% pyridine. The following conditions were used for the solid phase synthesis of all polyamides. Deblocking with 20% piperidine in DMF for 5 minutes, activation with 0.5M HATU / DMF and 1.0M DIEA / DMF for 2 minutes, binding for 60 minutes, 5 with 5% acetic anhydride and 5% pyridine in DMF Capping for a minute, and finally washing with methanol. When the monomer was bound to imidazole and when imidazole itself was bound, the coupling cycle was used twice, and the other coupling was performed once.

  In a typical case, Fmocβ-alanine clear acid resin (200 mg, 0.10 mmol) was swollen with 3 mL DMF for 30 minutes in a 15 mL plastic centrifuge tube and loaded into the reaction column. Glass scintillation tubes containing monomers in DMF solvent were placed on the rack in order from front to back. In this way, synthesis was started while being managed by a computer using a well-known program. Before and after adding the solution, all lines were evacuated with argon.

After the synthesis by the peptide synthesizer was completed, the resin was washed with a methanol / dichloromethane mixed solvent (1/1, v / v) and dried in vacuum at room temperature. The resin was then placed in a 20 mL glass scintillation vessel, 5 mL dimethylaminopropylamine was added, and the solution was stirred at 55 ° C. overnight. The resin was separated by filtration through a celite plate and washed with a methanol / dichloromethane mixed solvent (1/1, v / v). The filtrate was acidified to pH 4-5 with 10% hydrochloric acid and the solvent was removed in vacuo. The residue was dissolved in 0.5 mL of N, N-dimethylformamide, and the insoluble material was removed by filtration. The obtained polyamide solution was purified by HPLC. After confirmation by ¹ H NMR and ESI or TOF mass spectrometry, the desired polyamide 1,2,3,4 was freeze-dried as white or pale yellow powder.

(Polyamide synthesis by Fmoc chemistry: artificial synthesis procedure)
Further steps were required to obtain polyamide 5-12 from amino or hydroxyl modified monomers. Polyamide is removed from Resin 5a-12a in the presence of dimethylaminopropylamine, purified by HPLC, and dried to give polyamide protected with t-butoxycarbonyl (Boc) or t-butyl, ¹ H NMR and Confirmed by ESI or TOF mass spectrometry.

After this, the product was stirred for 30 minutes under a nitrogen atmosphere at room temperature using 0.5 μL of a mixed solvent of trifluoroacetic acid / triisopropylsilane / trimethylsilane / water (91/3/3/3, v / v), and in vacuum The solvent was evaporated off with dichloromethane. Hydrochloric acid (0.5 mL, 5 M) was added to the residue, and the solvent was removed in vacuo using cold potassium hydroxide and wrap. The residue was separated and analyzed by HPLC and confirmed by ¹ H NMR and ESI or TOF mass spectrometry. Finally, it was freeze-dried to obtain the target polyamide 5-12 as a white or pale yellow powder.

  The procedure for producing the polyamide is shown in FIG. The eight types of X and Y used are shown on the right. The reagents at each synthesis stage are as follows. (i) 20% piperidine / DMF, (ii) FmocPyCOOH, HATU, DIEA, (iii) 5% acetic anhydride + 5% pyridine / DMF, (iv) 20% piperidine / DMF, (v) 1a, 2a, 5a, 6a , 9a, 10a is Fmoc-β-Ala-OH, HATU, DIEA, 3a, 4a is Fmoc-γ-Abu-COOH, HATU, DIEA, 7a is compound 17a, HATU, DIEA, 8a is compound 17b , HATU, DIEA, 11a for compound 23, HATU, DIEA, for 12a compound 24, HATU, DIEA, (vi) 5% acetic anhydride + 5% pyridine / DMF, (vii) 20% piperidine / DMF, (viii) FmocPyCOOH, HATU, DIEA, (ix) 5% acetic anhydride + 5% pyridine / DMF, (x) 20% piperidine / DMF, (xi) FmocImCOOH, HATU, DIEA, (xii) 5% acetic anhydride + 5% pyridine / DMF, (xiii) 20% piperidine / DMF, (xiv) Fmoc-γ-Abu-COOH, (xv) 5% acetic anhydride + 5% pyridine / DMF, (xvi) 20% piperidine / DMF, (xvii) FmocPyCOOH, HATU, DIEA , (Xviii) 5% acetic anhydride + 5% pyridine / DMF, (xix) 20% piperidine / DMF, (xx) FmocImCOOH, HATU, DIEA, (xxi) 5% acetic anhydride + 5% pyridine / DMF, (xxii) 20% Piperidi / DMF, (xxiii) 1a, 3a, 7a, 8a, 11a, 12a for Fmoc-β-Ala-OH, HATU, DIEA, 2a, 4a for Fmoc-γ-Abu-COOH, HATU, DIEA, 5a Is compound 17a, HATU, DIEA, 6a is compound 17b, HATU, DIEA, 9a is compound 23, HATU, DIEA, 10a is compound 24, HATU, DIEA, (xxiv) 5% acetic anhydride + 5% pyridine / DMF (Xxv) 20% piperidine / DMF, (xxvi) FmocImCOOH, HATU, DIEA, (xxvii) 5% acetic anhydride + 5% pyridine / DMF, (xxviii) 1-4 for N, N-dimethylaminopropylamine, 5 -12 for Boc- and t-butyl protection, 55 ° C, overnight, (xxiv) 5-12 for TFA / TMS / TIS / H2O (91/3/3/3, v / v).

  According to this synthesis method, the number of repetitions of Py and Im is arbitrary, and p, q, r, s, and t can be arbitrarily selected as described in claim 3. Further, the sequence of Py and Im and the substituent Z can be arbitrarily selected.

The analysis results of the produced polyamide 25-28 are shown below.
The properties of polyamide 25 are as follows.
Yield as white powder: 8.53%.
_{HPLC:. T R = 11.60 min} at a 98% min purity.
¹ H NMR (400 MHz, DMSO-d ₆ ): d 10.35 (s, 1H, aromatic NH), d 10.29 (s, 1H, aromatic NH), 10.18 (s, 1H, aromatic NH), 9.87 (s, 1H , aromatic NH), 9.83 (s, 1H, aromatic NH), 9.44 (s, 1H, aromatic NH), 8.04 (m, 2H, aliphatic NH), 7.80 (t, 1H, aliphatic NH), 7.87-7.84 (m , 2H, aliphatic NH), 7.45 (s, 1H, aromatic CH), 7.43 (s, 1H, aromatic CH), 7.36 (s, 1H, aromatic CH), 7.20 (m, 2H, aromatic CH), 7.11 (d , 4JH, H = 1.6 Hz, 1H, aromatic CH), 6.92 (m, 2H, aromatic CH), 6.62 (d, 4JH, H = 1.6 Hz, 1H, aromatic CH), 4.14 (dd, ³ J _{H, H} = 8.0 Hz, ³ J _{H, H} = 3.6 Hz, 1H, CH ₂ CH ₂ C H OH), 3.95 (s, 3H, NCH3), 3.94 (s, 3H, NCH ₃ ), 3.90 (s, 3H, NCH ₃ ), 3.80 (s, 3H, NCH ₃ ), 3.79 (s, 3H, NCH ₃ ), 3.76 (s, 3H, NCH ₃ ), 3.51 (m, 2H, NHC H ₂ ), 3.42 (m, 2H, NHC H ₂ ), 3.32 (m, 4H, 2NHC H ₂ ), 3.03 (q, 2H, NHC H ₂ ), 2.60 (t, ³ J _{H, H} = 6.8 Hz, 2H, COCH ₂ ), 2.46 (m, 2H, NMe ₂ C H ₂ ), 2.29 (t, ³ J _{H, H} = 6.8 Hz, 2H, COCH ₂ ), 2.15 (t, ³ J _{H, H} = 7.2 Hz, 2H, COC H ₂ ), 2.06 ( s, 6H, N (CH ₃ ) ₂ ), 1.97 (s, 1H, COC _{H 3), 1.72 (m,} 2H, CH 2 C H 2 CHOH), 1.48 (m, 2H, CH 2 C H 2 CH 2).
ESI-Mass: m / z calcd for C ₅₃ H ₇₁ N ₂₁ O ₁₂ : 1193.5591; found: 1194.5700 [M + H] ⁺ , 597.7964 [M + 2H] ²⁺ .

The properties of polyamide 26 are as follows.
Yield as white powder: 18.7%.
_{HPLC:. T R = 11.55 min} at a 98% min purity.
¹ H NMR (400 MHz, DMSO-d ₆ ): d 10.35 (s, 1H, aromatic NH), 10.29 (s, 1H, aromatic NH), 10.19 (s, 1H, aromatic NH), 9.88 (s, 1H, aromatic NH), 9.83 (s, 1H, aromatic NH), 9.42 (s, 1H, aromatic NH), 8.06-7.99 (m, 3H, aliphatic NH), 7.88 (m, 2H, aliphatic NH), 7.45 (s, 4JH, H = 2.0 Hz, 1H, aromatic CH), 7.43 (s, ⁴ J _{H, H} = 2.0 Hz, 1H, aromatic CH), 7.36 (s, ⁴ J _{H, H} = 2.0 Hz, 1H, aromatic CH) , 7.20 (m, 2H, aromatic CH), 7.10 (s, 1H, aromatic CH), 6.93 (m, 2H, aromatic CH), 6.64 (s, 1H, aromatic CH), 4.13 (m, 1H, CH ₂ CH ₂ C H OH), 3.95 (d, ⁴ J _{H, H} = 2.0 Hz, 3H, NCH ₃ ), 3.94 (d, ⁴ J _{H, H} = 2.0 Hz, 3H, NCH ₃ ), 3.89 (d, ⁴ J _{H, H} = 2.0 Hz, 3H, NCH ₃ ), 3.79 (s, 3H, NCH ₃ ), 3.78 (s, ⁴ J _{H, H} = 2.0 Hz, 3H, NCH ₃ ), 3.76 (s, ⁴ J _{H, H} = 1.6 Hz, 3H, NCH ₃ ), 3.50-3.32 (m, 8H, 4NHC H ₂ ), 3.05 (q, 2H, NHC H ₂ ), 2.59 (t, ³ J _{H, H} = 5.6 Hz, 2H, COCH ₂ ), 2.43 (m, 2H, NMe ₂ CH ₂ ), 2.28 (m, 4H, 2COCH ₂ ), 2.07 (s, 6H, N (CH ₃ ) ₂ ), 1.97 (s, 1H, COCH ₃ ), 1.74 (m, 2H, CH ₂ C H ₂ CHOH), 1.57 (m, 2H, CH ₂ C H ₂ CH ₂ ).
ESI-Mass: m / z calcd for C ₅₃ H ₇₁ N ₂₁ O ₁₂ : 1193.5591; found: 1194.5987 [M + H] ⁺ , 597.7986 [M + 2H] ²⁺ .

The properties of polyamide 27 are as follows.
Yield as white powder: 20.5%.
_{HPLC:. T R = 9.95 min} at a 98% min purity.
¹ H NMR (400 MHz, DMSO-d ₆ ): d 10.35 (s, 1H, aromatic NH), 10.29 (s, 1H, aromatic NH), 10.07 (s, 1H, aromatic NH), 9.91 (s, 2H, aromatic NH), 9.85 (s, 1H, aromatic NH), 8.17 (t, 1H, aliphatic NH), 8.07 (t, 1H, aliphatic NH), 8.00 (t, 1H, aliphatic NH), 7.96 (t, 1H, aliphatic NH), 7.87 (t, 1H, aliphatic NH), 7.47 (s, 1H, aromatic CH), 7.44 (s, 1H, aromatic CH), 7.36 (s, 1H, aromatic CH), 7.21 (m, 2H, aromatic CH), 7.16 (d, ⁴ J _{H, H} = 1.6 Hz, 1H, aromatic CH), 6.98 (d, ⁴ J _{H, H} = 1.6 Hz, 1H, aromatic CH), 6.93 (d, ⁴ J _{H, H} = 1.6 Hz, 1H, aromatic CH), 6.64 (d, ⁴ J _{H, H} = 1.6 Hz, 1H, aromatic CH), 4.18 (m, 1H, CH ₂ CH ₂ C H NH ₂ ), 3.95 (s, 3H, NCH ₃ ), 3.94 (s, 3H, NCH ₃ ), 3.90 (s, 3H, NCH ₃ ), 3.80 (s, 6H, 2NCH ₃ ), 3.76 (s, 3H, NCH ₃ ), 3.51-3.31 ( m, 10H, 5NHC H ₂ ), 3.07 (m, 2H, NHC H ₂ ), 2.59 (m, 4H, NMe ₂ C H ₂ + NHC H ₂ ), 2.43 (s, 6H, NMe ₂ ), 2.31 (t, ³ J _{H, H} = 6.8 Hz, 2H, COCH ₂ ), 1.97 (s, 3H, COCH ₃ ), 1.80 & 1.97 (m, 2H, CH ₂ C H ₂ CHNH ₂ ), 1.63 (m, 2H, CH ₂ C H ₂ CH ₂ ).
ESI-Mass: m / z calcd for C ₅₃ H ₇₂ N ₂₂ O ₁₁ : 1192.5751; found: 1193.7769 [M + H] ⁺ , 597.7783 [M + 2H] ²⁺ .

The properties of polyamide 28 are as follows.
Yield as white powder: 9.80%.
_{HPLC:. T R = 9.93 min} at a 98% min purity.
¹ H NMR (400 MHz, DMSO-d ₆ ): d 10.34 (s, 1H, aromatic NH), 10.29 (s, 1H, aromatic NH), 10.15 (s, 1H, aromatic NH), 9.90 (s, 2H, aromatic NH), 9.83 (s, 1H, aromatic NH), 8.06 (m, 2H, aliphatic NH), 7.99 (t, 1H, aliphatic NH), 7.87 (m, 2H, aliphatic NH), 7.45 (s, 1H, aromatic CH), 7.43 (s, 1H, aromatic CH), 7.36 (s, 1H, aromatic CH), 7.20 (br, 2H, aromatic CH), 7.11 (s, 1H, aromatic CH), 6.94 (s, 1H, aromatic CH), 6.92 (s, 1H, aromatic CH), 6.62 (s, 1H, aromatic CH), 4.19 (m, 1H, CH ₂ CH ₂ CHNH ₂ ), 3.94 (s, 6H, 2NCH ₃ ), 3.90 ( s, 3H, NCH ₃ ), 3.79 (s, 6H, 2NCH ₃ ), 3.76 (s, 3H, NCH ₃ ), 3.51-3.30 (m, 10H, 5NHC H ₂ ), 3.03 (m, 2H, NHC H ₂ ), 2.59 (m, 2H, NMe ₂ C H ₂ ), 2.28 (t, ³ J _{H, H} = 6.8 Hz, 2H, COCH ₂ ), 2.19 (t, ³ J _{H, H} = 6.8 Hz, 2H, COCH ₂ ), 2.10 (s, 6H, NMe ₂ ), 1.97 (s, 3H, COCH ₃ ), 1.60 & 0.98 (m, 2H, CH ₂ C H ₂ CHNH ₂ ), 1.50 (m, 2H, CH ₂ C H ₂ CH ₂ ).
ESI-Mass: m / z calcd for C ₅₃ H ₇₂ N ₂₂ O ₁₁ : 1192.5751; found: 1193.8657 [M + H] ⁺ , 597.6786 [M + 2H] ²⁺ .

(Surface plasmon resonance test)
All surface plasmon resonance measurements were performed at 25 ° C. using a Biacore X instrument. Biotinylated hairpin DNA: 5'-biotin-GGCCGATG N 'GCATGCTATAGCATGC N CATCGGCC-3' (N ・ N ': T ・ A, A ・ T, G ・ C, C ・ G) is obtained from proligo It was. Streptavidin functionalized SA sensor chip was purchased from Biacore. The hairpin DNA was fixed to the sensor chip, the binding curve of the polyamide to the hairpin DNA, and the data analysis were performed as follows.

(Immobilization of biotinylated DNA)
After mounting the streptavidin-coated SA sensor chip on the device, wash it three times with HBS-EP buffer, standardize with Biacore standardization solution (40% glycerol aqueous solution), and buffer for about 10 minutes until the baseline is stable The liquid was allowed to flow at a rate of 10 μL / min. Thereafter, the sensor chip was conditioned 3-5 times with a 1 M sodium chloride / 50 mM sodium hydroxide solution at a flow rate of 10 μL / min in order to remove unbound streptavidin from the sensor surface. The buffer was then run for 10 minutes to remove the sodium hydroxide until the baseline was stable. Similarly, 20 μL of buffer solution was flowed three times at the same flow rate to wash the piping.
Before immobilizing DNA on the sensor chip, 100 μL of 5 ′ biotinylated DNA buffer solution was heated at 85 ° C. for 2 minutes without adding dimethyl sulfoxide, and slowly restored to room temperature for restoration. Thereafter, the DNA was immobilized at a selected flow cell by injecting an appropriate amount of DNA solution at a flow rate of 2 μL / min until a predetermined immobilization level was reached. After completion of DNA injection, the buffer continued to flow through the cell until the sensitivity of the sensor was constant. A flow cell without immobilized DNA was used as a basis for the matrix effect.

(General procedure for surface plasmon resonance measurement)
Polyamide solutions of various concentrations were prepared by diluting HBS-EP buffer containing 0.1% dimethyl sulfoxide to stock solutions (10 mM) using DMSO as a solvent. The concentration was confirmed by ultraviolet absorption at 294 nm. Unless otherwise specified, the above buffer solution was used for the following measurements, and this buffer solution or 10 mM glycine-HCl solution (pH = 1.5) was used as a regeneration solution.
Typically, after the baseline was stable, buffer was injected as a blank at a flow rate of 5 μL / min. Samples were then run under the same conditions at concentrations ranging from 5 nM to 5 μM. In order to avoid the influence of the previous measurement, the samples were injected in order from light to dark. Using the BIA evaluation 4.1 program, the signal with respect to time was fitted to the mass transfer effect model, and dynamic analysis was performed.

(Calculation method of molecular model)
Minimization was performed using the Discover program (MSI, San Diego, USA) using CVFF force field parameters. The initial structure was based on the structure of IMPyPy-γ-PyPyPy-5′-d (CGCTAACAGGC) -3 ′ / 5′-d (GCCTGTTAGCG) -3 ′ complex determined by NMR. Aliphatic monomers were inserted into the polyamide using standard bond lengths and bond angles. Twenty-two sodium cations were placed in two equal parts of the OPO angle at a distance of 2.21 cm from the phosphorus atom. The resulting complex was applied to a 10 cm aqueous layer. The entire system was unconditionally minimized so that the RMS was 0.001 kcal / mol ^ or less.

Synthesis scheme of two new monomers. Polyamide synthesis scheme. Molecular model of the interaction of polyamide with hairpin DNA. (a) When W is (CH2) 3, (b) When W is γSO or γRO. Time change of surface plasmon resonance signal due to interaction between polyamide and hairpin DNA immobilized on sensor chip. Table of dissociation equilibrium constant (M) and selectivity of polyamide 1-12 with hairpin DNA. Table of dissociation equilibrium constant (M) and selectivity of polyamide 1,25-28 with hairpin DNA. Molecular model of the interaction between polyamide 5 with β / γSO pair and DNA (5'-GCATGC A / T CATCG-3 '/ 5'-CGATG A / T GCATGC-3').

Claims (9)

An optically active 2-t-butyloxy- (3 + n)-(9-fluorenylmethoxy) carbonylaminoalkylcarboxylic acid of the S configuration or R configuration represented by the general formula (1).
(In the formula, C ^* is an optically active carbon atom, which takes S configuration or R configuration, n represents a natural number, and Fmoc represents a fluorenylmethoxycarbonyl group. A hydrogen atom bonded to a carbon atom of the main chain. (The hydrogen atom in the molecule may be replaced with a deuterium atom.)   An optically active 2-t-butyloxy-4- (9-fluorenylmethoxy) carbonylaminoalkylcarboxylic acid of S configuration or R configuration, wherein n = 1 in the general formula (1). Starting from an optically active (3 + n) -amino-2-hydroxyalkyl carboxylic acid of S configuration or R configuration,
(a) The starting material is reacted with (9-fluorenylmethoxy) carbonyl succinimide in a mixture of water and 1,2-dimethoxyethane in the presence of a small amount of sodium bicarbonate to give optically active (3 + n)- [(9-Fluorenylmethoxy) carbonylamino] -2-hydroxyalkylcarboxylic acid was obtained,
(b) Suspend the alkyl carboxylic acid in dichloromethane, add excess isobutene at room temperature and continue the reaction until a clear solution is obtained, 2-t-butoxy- (3 + n)-(9-flux (Oleenylmethoxy) carbonylaminoalkylcarboxylic acid t-butyl ester
(c) A highly active silyl substitution reagent, trimethylsilyl trifluoromethanesulfonic acid (TMSOTf), is added to the butyl ester and reacted.
A method for producing an optically active 2-t-butyloxy- (3 + n)-(9-fluorenylmethoxy) carbonylaminoalkylcarboxylic acid having an S configuration or an R configuration represented by the general formula (1).   The method for producing an optically active 2-t-butyloxy-4- (9-fluorenylmethoxy) carbonylaminoalkylcarboxylic acid having S configuration or R configuration according to claim 3, wherein n = 1. A hairpin type polyamide represented by the general formula (2).
(In the formula, X and Y are substituted or different from each other selected from β-alanine, diaminoalkylcarboxylic acid, optically active α, ωaminoalkylcarboxylic acid, and optically active αhydroxyl-ωaminoalkylcarboxylic acid. And W represents (CH ₂ ) _r , β-alanine, diaminoalkylcarboxylic acid, optically active α, ωaminoalkylcarboxylic acid, optically active αhydroxyl-ωaminoalkyl Substituents selected from among carboxylic acids, p, q, r, s and t each represent an independent natural number, and Z represents a terminal diamino group or an alkylation reaction site. Alternatively, a part may be replaced with an imidazole ring.) In general formula (2), r = 3, p = 2, q = 1, s = 2, t = 1, including three pyrrole rings and three imidazole rings, and W is (CH ₂ ) ₃ The hairpin type polyamide according to claim 5, represented by the following structural formula (3), wherein Z is a propyldiamine cation group.
(In the formula, X and Y are substituents selected from β-alanine, diaminobutyric acid, optically active α, γdiaminobutyric acid, and optically active αhydroxyl-γaminobutyric acid, which are equal to or different from each other.) In general formula (2), r = 3, p = 2, q = 1, s = 2, t = 1, including three pyrrole rings and three imidazole rings, and W is optically active α, γ diaminobutyric acid The hairpin-type polyamide according to claim 5, which is a substituent selected from optically active α-hydroxyl-γ-aminobutyric acid, and Z is a propyldiamine cation group and represented by the following structural formula (4).
(In the formula, X, Y and W are substituents selected from β alanine, diaminobutyric acid, optically active α, γ diaminobutyric acid and optically active αhydroxyl-γaminobutyric acid, which are equal to or different from each other. )   A method for recognizing DNA using the polyamide according to claim 5, 6 or 7. A method for recognizing an AT base pair or a TA base pair of DNA using the polyamide according to claim 5, 6 or 7.