JP5812478B2 - Anticancer drug-binding nucleic acid aptamer and use thereof - Google Patents

Description

  The present invention relates to a novel anticancer agent-binding nucleic acid aptamer, an anticancer agent containing the same, and a novel compound.

  Camptothecins such as irinotecan and topotecan have a wide anticancer spectrum and are therefore used as one of very effective cancer therapeutic agents. However, side effects such as myelosuppression and hemorrhagic cystitis have been reported, and medication requires special attention. If a safe dosing method with few side effects is developed for camptothecins effective against various cancer diseases, the gospel is brought to many patients suffering from cancer.

In order to reduce side effects, it is considered effective to deliver the minimum amount of camptothecins to the disease site. For this purpose, a DDS function for specifically delivering a drug to the disease site can be bound to the camptothecin derivative, Moreover, it is necessary to retain the activity of the derivative. As the method, there is a method of covalently connecting a polymer to a camptothecin derivative. However, this method has a problem that it is difficult to smoothly perform drug release (release of a drug when bound to a diseased cell tissue).
On the other hand, although the development of molecular target drugs using nucleic acid aptamers has been promoted (Non-patent Document 1), no nucleic acid aptamers that can directly bind to anticancer agents have been reported.

Shin Miyagawa, Masatoshi Fujiwara, Michihisa Nishiyama: Development of molecularly targeted drugs using RNA aptamers. DDS 23: 534-543. 2008.

  An object of the present invention is to provide a nucleic acid aptamer that specifically recognizes and binds to camptothecins.

  The present inventor has intensively studied to solve the above problems. As a result, it was found that a nucleic acid aptamer containing a specific sequence such as SEQ ID NO: 1 or 2 specifically binds to camptothecins, thereby finding that it can be used for measurement of pharmacological effects and pharmacokinetics of anticancer agents and camptothecins. Thus, the present invention has been completed.

That is, the present invention provides a camptothecin binding agent comprising a nucleic acid aptamer containing any of the following sequences.
(I) GGGNGGGNGGGNGGG (SEQ ID NO: 1)
(Ii) GGGNGGGNGGGNNGGG (SEQ ID NO: 2)
Here, N represents A, T, G, or C.
The sequence containing (i) or (ii) includes the sequence of base numbers 21 to 50 of any one of SEQ ID NOs: 6 to 9 and 12 to 18, the sequence of base numbers 21 to 51 of SEQ ID NO: 10, SEQ ID NO: 11 or It is a sequence of 20 base numbers 21 to 49, a sequence of base numbers 21 to 52 of SEQ ID NO: 19, a sequence of base numbers 14 to 40 of SEQ ID NO: 23, or a sequence of base numbers 21 to 54 of SEQ ID NO: 21 More preferred.

The nucleic acid aptamer may contain a modified base. That is, the present invention also provides the sequence of base numbers 21 to 50 of any of SEQ ID NOs: 28 to 32, 35 to 43, and 45 to 59, the sequence of base numbers 21 to 51 of SEQ ID NO: 44, or the base of SEQ ID NO: 60 Provided is a camptothecin binding agent comprising a nucleic acid aptamer comprising the sequences of Nos. 21 to 48. In addition, the modification T in these sequences indicates a residue of a compound represented by the following formula (I).

The present invention also provides a compound represented by the following formula (I) or a salt thereof. Examples of the salt include metal salts.
This compound is a derivative of thymidine triphosphate. Therefore, it can be used for production of modified polynucleotides.

The present invention also provides a compound represented by the following formula (II) or (III) or a salt thereof. Examples of the salt include acid addition salts.
These are derivatives of camptothecin known as anticancer agents. Therefore, these compounds can be used as anticancer agents alone or in combination with a pharmaceutically acceptable carrier. The dosage, administration route, dosage form, carrier to be combined, etc. can be determined in accordance with the camptothecin derivative known as an anticancer agent.

In the present invention, the camptothecins are not limited to camptothecin, and any camptothecin may be used, as long as it includes a camptothecin skeleton. ), (III
).

The nucleic acid aptamer is preferably DNA, more preferably single-stranded DNA. The nucleic acid aptamer may be modified. For example, phosphorothioate or phosphorodithioate modifications can be made to increase the stability of the nucleic acid aptamer. Further, it may be labeled with a fluorescent substance or the like.
The length of the nucleic acid aptamer is preferably 15 to 100 bases, more preferably 20 to 60 bases.

Since the nucleic acid aptamer newly developed in the present invention can specifically recognize and bind camptothecins, as a molecular probe for drug detection or a drug carrier molecule of DDS, It is expected to be applied to validation studies such as pharmacokinetics and cancer treatment by DDS.
Nucleic acids can be (1) able to suppress biological rejection, (2) can be easily linked to other nucleic acid aptamers or antibodies that specifically recognize specific molecules on the cell surface, etc. (3) linked between nucleic acids In this case, it is considered that the method using a polymer is advantageous in that an intelligent function such as drag / release can be easily provided.

The figure which shows the arrangement | sequence of the camptothecins binding nucleic acid aptamer selected in Example 2. FIG. The consensus sequences are shown in groups. The numbers in parentheses indicate the sequence numbers. The underlined part is the primer sequence.

The present invention is described in detail below.
One embodiment of the camptothecin binding agent of the present invention includes the following sequence (i) or (ii):
(I) GGGNGGGNGGGNGGG (SEQ ID NO: 1)
(Ii) GGGNGGGNGGGNNGGG (SEQ ID NO: 2)
Here, N represents A, T, G, or C.

  Specifically, the sequence of nucleotide numbers 21 to 50 of any one of SEQ ID NOs: 6 to 9 and 12 to 18, the sequence of nucleotide numbers 21 to 51 of SEQ ID NO: 10, the sequence of nucleotide numbers 21 to 49 of SEQ ID NO: 11 or 20 , A sequence of base numbers 21 to 52 of SEQ ID NO: 19, a sequence of base numbers 14 to 40 of SEQ ID NO: 23, or a sequence of base numbers 21 to 54 of SEQ ID NO: 21. As long as the ability to bind to at least one camptothecin is maintained, 1 to several, for example, 1, 2 or 3 bases may be substituted, deleted, inserted, etc. in these sequences. Further, any sequence of any length may be added to the 5 'side and / or 3' side as long as the ability to bind to at least one camptothecin is maintained.

In addition, other embodiments of the camptothecin binding agent of the present invention include the sequence of base numbers 21 to 50 of any one of SEQ ID NOs: 28 to 32, 35 to 43, and 45 to 59, and base number 2 of SEQ ID NO: 44.
It may be a nucleic acid aptamer containing a modified base comprising the sequence of 1 to 51 or the sequence of base numbers 21 to 48 of SEQ ID NO: 60. As long as the ability to bind to at least one camptothecin is maintained, 1 to several, for example, 1, 2 or 3 bases may be substituted, deleted, inserted, etc. in these sequences. Further, any sequence of any length may be added to the 5 ′ side and / or the 3 ′ side as long as the ability to bind to at least one kind of camptothecins is maintained.

Moreover, since the following consensus can be extracted from FIG. 1, the nucleic acid aptamer containing the following sequences can also be used as a camptothecin binding agent.
ACGtAGGGtGGtCAtAGtAtC
tGGGAAACGGGtGGttt
AAAGGtXXXXXXXXXXXXXGttXC
AtCCCGtt… tAACC
GCCttAA
GtXAtGGCG… CCtXtGXA
CGCCAtCTTC
AAAAA ... CttttC
AGGGGXAtXXXCtA
Here, X represents an arbitrary base (preferably A, G, C or t), and t is a base part of the compound of the formula (I).

  The camptothecin binding agent of the present invention can be used as a molecular probe for detecting camptothecins. For example, a nucleic acid aptamer can be labeled with a fluorescent dye or the like to form a complex with camptothecins, and the pharmacokinetics of camptothecins can be traced.

Moreover, the complex of the nucleic acid aptamer and camptothecins of the present invention can be used as an anticancer agent. Preferably, for DDS (drug delivery system), it is preferable to bind an aptamer with a substance for specifically accumulating camptothecins in a target cancer tissue. The substance for specifically accumulating camptothecins in the target cancer tissue is preferably a substance that specifically binds to a protein or sugar chain that appears on the cell surface of the target cancer tissue, such as an antibody protein or cell surface. Other nucleic acid aptamers that specifically recognize these specific molecules are more preferred.
Techniques for binding such substances to aptamers are known. For example, another sequence (a sequence that does not affect binding) is added to a sequence involved in binding to camptothecins, and biotin is bound thereto. On the other hand, the antibody can be labeled with avidin, and the antibody and aptamer can be bound to form an aptamer-camptothecins-antibody complex.

  The following examples illustrate the present invention more specifically. However, the present invention is not limited to the following examples.

Synthesis of Camptothecin Derivatives Camptothecin derivative 1 (Compound e) and Camptothecin derivative 2 (Compound c) were synthesized by the following procedure.

(S)-(+)-Camptothecin (200 mg, 5.7 × 10 ⁻⁴ mol, 1.0 eq) was dissolved in 75% H ₂ SO ₄ (5 ml) under ice cooling. FeSO ₄ · 7H ₂ O (250 mg, 9 × 10 ⁻⁴ mol, 1.58 eq) was dissolved in water (1 ml) and added to the Camptothecin solution under ice cooling. ClCH ₂ CHO (3.2 ml) was added to the Camptothecin solution under an ice bath. After sufficiently stirring the solution, 30% H ₂ O ₂ (1 ml) was added dropwise under ice cooling, and the mixture was stirred for 5 min under ice cooling and then stirred at room temperature for 1.5 h. Cold water was added to the reaction solution under ice cooling and suspended. The reaction solution was suction filtered using Celite, and the residue was DMF.
And then distilled off under reduced pressure. The residue was suspended in a chloroform: hexane = 7: 3 solution and subjected to suction filtration to obtain a yellow powdery compound 1. The filtrate was purified by silica gel chromatography (Silica gel 60, 70-230mesh, 0-10% methanol / chloroform) to obtain compound a.
Yield: 164 mg Yield: 72%
ESI-MS (POS) Measured value: 419.2 Calculated value: 419.1 [M + Na] (m / z)
¹ H NMR (DMSO) δ 8.42 (1H, d) 8.27 (1H, d) 7.94 (1H, t) 7.83 (1H, t) 6.56 (1H, s)
5.45-5.39 (6H, m) 1.92 (2H, m) 0.89 (3H, t)

Compound a (300 mg, 7.56 × 10 ⁻⁴ mol, 1.0 eq) was vacuum-dried and dissolved in dehydrated DMF (20 ml). 4- (tert-Butoxycarbonylamino) piperidine (757 mg, 3.78 × 10 ⁻³ mol, 5.0 eq) was dissolved in dehydrated DMF (15 ml), dehydrated Toluene (35 ml) was added, added to the reaction solution, and stirred for 2 h. The solution was evaporated under reduced pressure, dissolved in CHCl ₃ and washed with saturated brine, and the organic phase was dried over MgSO ₄ . The solution was evaporated under reduced pressure and purified by silica gel column chromatography (Fuji Silysia FL60D, chloroform → 70% ethyl acetate / hexane) to obtain Compound b.
Yield: 76.3 mg Yield: 18%
ESI-MS (POS) Measured Value: 583.3 Calculated Value: 583.3 [M + Na] (m / z)
¹ H NMR (CDCl ₃ ) δ 8.31 (1H, d) 8.19 (1H, d) 7.80 (1H, t) 7.65-7.60 (2H, m)
5.77 (ABquartet, 2H) 5.38 (2H, s) 4.06 (2H, s) 3.56 (1H, m)
2.97 (2H, m) 2.34 (2H, t) 2.08-1.85 (4H, m) 1.75 (2H, m)
1.43 (9H, m) 1.06 (3H, t)

Compound b (150 mg, 2.68 × 10 ⁻⁴ mol) was dissolved in 10% TFA / CH ₂ Cl ₂ (18 ml) and stirred for 1.5 h. Excess TFA was removed by azeotropy with MeCN (10 ml × 8) to obtain compound c (Camptothecin derivative 2).
Yield: 130 mg (ditrifluoroacetate) Yield: 71%
ESI-MS (POS) Measured value: 461.4 Calculated value: 461.2 [M + H] (m / z)
¹ H NMR (CD ₃ OD) δ 8.36 (1H, d) 8.14 (1H, d) 7.85 (1H, t) 7.72 (1H, t)
7.59 (1H, s) 5.59 (ABquartet, 2H) 5.43 (2H, s) 4.59 (2H, s)
3.40 (1H, m) 2.90 (2H, m) 2.15 (2H, m) 1.96-1.91 (4H, m)
1.41-1.28 (2H, m) 1.02 (3H, t)

Compound c (120 mg, 1.74 × 10 ⁻⁴ mol, 1.0 eq) was lyophilized and dehydrated DMF (4 ml) was added and dissolved. Dissolve O- [2- (Boc-amino) -ethyl] -O '-[2- (diglycolyl-amino) ethyl] decaethylene glycol (216 mg, 2.83 × 10 ^-4 mol, 1.6 eq) in dehydrated DMF (4 ml) HBTU (148.8 mg, 3.92 × 10 ^-4 mol, 2.3 eq), HOBtH ₂ O (60 mg, 3.91 × 10 ^-4 mol, 2.2 eq), DIPEA (90.3 μl, 5.20 × 10 ^-4 mol, 3.0 eq) was added in turn with good stirring to the solution of compound c. The reaction was allowed to proceed for 2 h at room temperature. The reaction mixture was evaporated under reduced pressure, dissolved in CHCl ₃ and washed with saturated brine. MgSO ₄ was added to the organic phase for drying, suction filtration was performed, and the residue was distilled off under reduced pressure.
Yield: 302mg (crude product)
ESI-MS (POS) Measured value: 1224.9 Calculated value: 1225.6 [M + Na] (m / z)
¹ H NMR (CDCl ₃ ) δ 8.39 (1H, d) 8.23 (1H, d) 7.82 (1H, t) 7.73 (1H, m) 7.40 (4H, m)
5.61 (2H, m)
5.29 (1H, m) 5.11 (1H, s) 4.05 (4H, m) 3.64 (40H, m) 3.30 (5H, m)
3.13 (6H, m)
2.97 (1H, d) 2.82 (1H, m) 2.00 (2H, m) 1.91-1.84 (4H, m)
1.49 (9H, m) 1.06 (3H, t)

Compound d (117 mg) was dissolved in 10% TFA / CH ₂ Cl ₂ (8 ml) and stirred for 2 h. Excess TFA was removed by azeotropy with MeCN (10 ml × 8), followed by purification by HPLC to obtain compound e (Camptothecin derivative 1).
Yield: 79.1mg Yield: 88% (from b)
ESI-MS (POS) Measured value: 1103.3 Calculated value: 1103.6 [M + H] (m / z)
¹ H NMR (CDCl ₃ ) δ 8.40 (1H, d) 8.21 (1H, d) 7.83 (1H, t) 7.71 (1H, t)
7.64 (1H, s) 5.75 (ABquartet, 2H) 5.49 (2H, s) 4.10 (4H, s)
3.82 (2H, s) 3.71 (2H, m) 3.64 (40H, m) 3.50 (3H, m)
3.19-3.13 (4H, m) 2.02-1.86 (10H, m) 1.76 (2H, m)
1.05 (3H, t)

Example 1
Selection (Natural DNA) targeting Camptothecin derivative 1
<Buffer used in Selection>
・ LC-AT
(10 mM phosphoric acid, 138 mM NaCl, 2.7 mM KCl, 2.5 mM MgCl ₂ , 0.05% Tween # 20) pH 7.4
・ LC-A
(10 mM phosphoric acid, 138 mM NaCl, 2.7 mM KCl, 2.5 mM MgCl ₂ ) pH 7.4
・ LC-B
(7M Urea, 10 mM phosphoric acid, 138 mM NaCl, 2.7 mM KCl, 2.5 mM MgCl ₂ ) pH 7.4
・ LC-B2
(0.2 mM Camptothecin derivative 2, 10 mM phosphoric acid, 138 mM NaCl, 2.7 mM KCl, 2.5 mM MgCl ₂ ) pH 7.4
・ SC-N
(0.05M Na ₂ HPO ₄ 0.05M NaH ₂ PO ₄ , 0.1% NaN ₃ ) pH 7.0
・ 1 × CB-NO
(0.2M NaHCO ₃ , 0.5M NaCl) pH8.3
・ Washing Buffer A
(0.5M ethanolamine, 0.5M NaCl) pH8.3
・ Washing Buffer B
(0.1M acetic acid, 0.5M NaCl) pH4.0

<DNA used>
・ MRC # T1F (5'-end FAM label * This corresponds to Rnd # 70 at the time of fluorescent titration)
5'-GGTCAGCACGCTCCGGACTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGTGTCGCTGAGCCTGCCAAC-3 'SEQ ID NO: 3
・ MRC # P1F (5 'terminal FAM labeling)
5'-GGTCAGCACGCTCCGGACTT-3 'SEQ ID NO: 4
・ MRC # P2P (5 'terminal phosphorylation)
5'-GTTGGCAGGCTCAGCGACAC-3 'SEQ ID NO: 5
・ MRC # P1N
5'-GGTCAGCACGCTCCGGACTT-3 'SEQ ID NO: 4
・ MRC # P2N
5'-GTTGGCAGGCTCAGCGACAC-3 'SEQ ID NO: 5

<Preparation of affinity gel>
・ Preparation of gel for positive selection
Add 2ml x 3 1mM HCl to Hitrap NHS-activated HP Columns (GE HealthCare Life Sciences) and 1ml Camptothecin derivative 1 solution (dissolved in 1 x CB-NO, 2.5μmol, total 3.2ml)
Add hydrochloric acid to remove the remaining 2.2ml and repeat for 1h. Add 3ml of 1x CB-NO, then. Wash Buffer A was added in 2ml x 3, Wash Buffer B in 2ml x 3, Wash Buffer A in 2ml x 3, and left for 30min. Thereafter, 2 ml × 3 of washing buffer B, 2 ml × 3 of washing buffer A, and 2 ml × 3 of washing buffer B were added. 1 ml × 3 of 1 mM HCl was added and 2 ml × 2 of SC-N was added to preserve the gel.

・ Preparation of gel for negative selection
2 ml × 3 of 1 mM HCl was added to Hitrap NHS-activated HP Columns, 2 ml × 3 of Washing Buffer A, 2 ml × 3 of Washing Buffer B, and 2 ml × 3 of Washing Buffer A were added and allowed to stand for 30 min. Thereafter, 2 ml × 3 of washing buffer B, 2 ml × 3 of washing buffer A, and 2 ml × 3 of washing buffer B were added. 1 ml × 3 of 1 mM HCl was added and 2 ml × 2 of SC-N was added to preserve the gel.

<Screening by Affinity Chromatography>
1 × LC-AT (400 μl) of a chemically synthesized DNA library (180 pmol: 1st round) or a DNA library (100 pmol: 2nd round or later) prepared from the eluate showing the binding activity of the previous round
After UV measurement, the sample was refolded [94 ° C., 30 sec → 25 ° C. (0.5 ° C./min)]. The negative selection gel and the positive selection gel were substituted with Buffer 1 × LC-AT 400 μl × 4 times, and the DNA was incubated on the negative selection gel (37 ° C., 10 rpm, 1 h). Supernatant is collected, UV measurement is performed, and then incubated on a gel for positive selection (37 ° C,
10 rpm, 1 h). The supernatant was collected and UV was measured. The positive selection gel was replaced with Buffer and unbound DNA was removed with 400 μl × 4 times of LC-A. * After 9 rounds, add 400μl of LC-A and incubate (37 ℃, 10rpm, 5min) and remove the supernatant (x5 times)
400 μL of elution buffer (LC-B up to round 5 and LC-B2 up to round 6) was added to the positive selection gel to elute DNA bound to the positive selection gel (up to 3 rounds up to 3 Times, once after 6 rounds). The eluted solution was desalted with a spin column and freeze-dried, and then 162 μl of distilled water and 18 μl of 10 × KOD Dash Buffer were added to dissolve the DNA. PCR was performed using this solution.
The amount of DNA incubated on the gel for positive selection was measured by UV, and the amount of eluted DNA was measured by denaturing PAGE, and the progress of selection was confirmed from the ratio.

<Amplification of eluted DNA>
DNA showing binding activity eluted at 37 ° C. was amplified by performing polymerase chain reaction (PCR). Take a small sample after the reaction, add 0.1% BPB solution containing 7M Urea 3mM EDTA, denature (94 ℃, 5min), and react by gel electrophoresis (200V, 35min, 45 ℃) using 10% denaturing PAGE Confirmed progress. In PCR, MRC # P1F and MRC # P2P were used as primers, and KOD Dash DNA polymerase was used as an enzyme.

<Preparation of single-stranded DNA> ^{* 1}
The PCR reaction solution was lyophilized using a rotor bag and then dissolved in distilled water. 3M CH ₃ COONa was added and mixed lightly, EtOH was added, and ethanol precipitation was performed. After standing 10 minutes in the freezer
Centrifugation (12000 rpm, 15 min, 4 ° C.) was performed, the supernatant was removed, and the residue was collected in one microtube and freeze-dried. The residual double-stranded DNA was reacted with λexonuclease at 37 ° C. for 30 min. Collect a small amount of the solution after the reaction, add 0.1% BPB solution containing 7M Urea 3mM EDTA, denature (94 ° C, 5min), gel electrophoresis using 10% denaturing PAGE (200V, 35min, 45 ° C) The progress of the reaction was confirmed by staining with SYBR Gold.
The DNA solution made single-stranded by λexonuclease was ethanol precipitated. Single-stranded DN of residue
A was redissolved in distilled water and purified by denaturing PAGE (7M Urea) and used for the next round of libraries.

<Aptamer isolation and sequence analysis>
Aptamers were isolated from the enriched library by ordinary cloning methods. MRC # P1N and MRC # P1N were used as primers for PCR preparation from a library in which DNA fragments to be inserted into vectors were concentrated. PT7Blue (Takara Bio) was used as the primer vector and Competent high E.coil DH5α (Toyobo) was used as the competent cell. A plasmid was taken out from each generated colony, and sequence analysis was performed with a sequencer.

<Gel affinity assay>
The obtained DNA (60 pmol) was dissolved in 70 μl of 1 × LC-AT and annealed [94 ° C., 30 sec → 25 ° C. (0.5 ° C./min)]. Of this, 5 μl was dispensed into another microtube (dispensing 1). 400 μl of 1 × LC-AT was added to each of the negative selection gel and the positive selection gel, vortexed and centrifuged, and the supernatant was removed. The buffer was replaced by repeating this operation 4 times. Each gel was incubated with 30 μl of the DNA solution and shaken at 37 ° C., 1200 rpm for 1 h. Vortex and centrifuge and collect 5 μl of supernatant. Add 0.1% BPB solution containing 7M Urea 3mM EDTA to the sample before incubation (Dispensing 1), the supernatant sample for negative selection gel, and the supernatant sample for positive selection gel, and denature (94 ℃, 5min). Perform gel electrophoresis (200V, 35min, 45 ° C) using 10% denaturing PAGE,
By introducing the band intensity into the following equation, the binding ratio to the target molecule was calculated.

Binding ratio (%) = {(NEG-POS) / NI} × 100
Here, NEG is the supernatant of the negative selection gel, POS is the supernatant of the positive selection gel, and NI is the amount of each DNA in the solution (injection 1) before incubation.

After 11 rounds of selection, the sequence obtained was analyzed and the results shown in Table 1 were obtained.

Next, among the sequences obtained in Table 1, Apt4 # 70, Apt14 # 70, Apt22 # 69, Apt4 # 40, Apt4
For # 20 (core sequence) and Rnd # 70 (library), binding to camptothecins was examined by fluorescence titration.

<Protocol for fluorescence titration>
-Preparation of DNA Each DNA dissolved in sterilized water was mixed with 5 × Selection buffer, and a DNA solution dissolved in 1 × Selection buffer was prepared with a final.

・ Preparation of compound solution Dissolve each compound in sterilized water (if it cannot be dissolved, filter with Millipore filter, use the filtrate) and mix with the above 5x Selection buffer. A 2 μM solution of the dissolved compound was prepared.

The structure of the Acridon derivative used is as follows.

Measurement procedure DNA prepared at each concentration was annealed. [94 ℃, 30sec → 25 ℃ (0.5 ℃ / min)]
2. 50 μL of the compound solution prepared to 2 μM was placed in a microtube in advance, 50 μL of each concentration of DNA was added thereto, and vortexing and centrifugation were performed three times. (Incubation)
3. Fluorescence was measured from the low concentration.

As a result, as shown in Table 9, Apt4 # 70, Apt14 # 70, Apt22 # 69 and Apt4 # 40 showed specific binding to camptothecins, but Apt4 # 20 and Rnd # 70 did not bind. It was.

Example 2: Screening of DNA aptamers containing modified bases First, modified bases were synthesized.

Synthesis of KS5
Dissolve 6-chloro-9H-purine (300 mg, 1.94 mmol, FW154.56) in butanol: water-6: 1 mixture (7 mL), and add ethane-1,2diamine (1.3 mL, 0.19 mmol, FW60.10). In addition, a reflux tube was installed, and the mixture was heated to 80 ° C. with an oil bath and stirred for 2 hours. After distilling off the reaction solution under reduced pressure, the residue was dissolved in water (1 mL),
Further, methanol (1 mL) and butanol (6 mL) were added and mixed well, and then concentrated by distillation under reduced pressure to precipitate crystals. This was suction filtered to obtain the intended product 1.

Yield 306 mg Yield 88.5%

¹ H NMR (300MHz D ₂ O)
δ3.11 (2H, t-, NH- CH 2- C H 2 -)
δ3.69 (2H, t, -NH- C H 2- CH 2 -)
δ7.94 (H, s, = NC H = N-)
δ8.05 (H, s, -HN-C H = N-)

ESI-MS (POS) m / z [Attribution]
Found: 179.1 Calc179.1 [(M + H) ⁺ ]

Synthesis of KS6
(E) -5- (2-carboxyrinyl) -2-deoxyuridine (335.3 μmol, 100 mg, FW298.25) was dissolved in dehydrated DMF (2 mL), PyBOP (403.5 μmol, 210 mg, FW520.39), HOBt (423.6 μmol) , 65 mg, FW153.44) and DIPEA (3.44 mmol, 600 μL, FW129.25 d = 0.742 g / mL) were added and lightly mixed. Further, a suspension obtained by suspending vacuum-dried 1 (404.0 μmol, 72 mg, FW 178.2) in dehydrated DMF (24 mL) was added, and the mixture was stirred for 1.5 hours. After completion of the reaction, the reaction mixture was concentrated by distillation under reduced pressure and filtered with suction. After concentrating the filtrate, chloroform was added to precipitate a precipitate, and suction filtration was performed to obtain the target product 2.

Yield 204.4mg Yield 87.7%

¹ H NMR (300MHz DMSO)
δ8.28 (H, s, -C = C H -N-)
δ8.19 (H, s, = NC H = N-)
δ8.09 (H, s, -N = C H -NH-)
δ7.10 (1H, s, -CC H = CH-)
δ7.03 (1H, s, -CH = C H -C-)
δ6.15 (1H, t, -OC H -CH _2- )
δ5.27 (1H, m, -OC H - CHOH-)
δ5.17 (1H, m, -CH-C H OH-CH _2- )
δ4.26 (1H, m, -OH-C H ₂ -C-)
δ3.80 (1H, m, -OH-C H ₂ -C-)
δ2.16 (2H, m, -CH-C H _2- CHOH-)

ESI-MS (POS) m / z [Attribution]
Found: 480.8 Calc481.2 [(M + Na) ⁺ ]

Synthesis of KS7
Vacuum-dried 2 (50 mg, 109.1 μmol, FW 458.4) was azeotroped twice with dehydrated pyridine (30 mL), dissolved in dehydrated trimethyl phosphate (30 mL), and phosphorous choride (1.61 mmol, 150 μL, under ice cooling) FW153.33, d = 1.645 g / mL) was added, and the mixture was stirred for 4 hours. After completion of the reaction, cold water (15 mL) and TEA (4.91 mmol, 680 μL, FW101.19, d = 0.73 g / mL) were added, and the mixture was stirred for 10 minutes to quench. The mixture was concentrated by distillation under reduced pressure, acetonitrile and diethyl ether were added, a precipitate was precipitated, and the filtrate was collected as a target product by suction filtration. Then, it melt | dissolved in a small amount of distilled water and refine | purified with the anion exchange column.
The yield was calculated using a molar extinction coefficient of 2 (ε _{260 nm} = 22200 mol ⁻¹ Lcm ⁻¹ ).

Yield 970OD Yield 40.0%

ESI-MS (NEG) m / z [Attribution]
Found: 536.7 Calc: 537.4 [(MH) ^- ]

Synthesis of KS8
Vacuum-dried 3 (78.89 μmol, 42.47 mg, FW538.1) was azeotroped twice with dehydrated pyridine (1.5 mL), dissolved in dehydrated DMF (600 μL), and TEA (4526.6 μmol, 73 μL, FW101.19, d = 0.73g / mL) and stirred for 5 minutes, Imidzole (318.7μmol, 21.7mg, FW68.08), Triphenyl phosphine (126.2μmol, 33.1mg, FW262.29), 2,2'-Dipridyl Disulfide ( 128.4 μmol, 28.3 mg, FW220.32) was added, and the mixture was stirred at room temperature for 3 hours. After completion of the reaction, the reaction solution was added to a mixed solution of sodium perchlorate and dehydrated aceton and allowed to stand at 4 ° C. for 30 minutes. The precipitate was washed with dehydrated aceton and obtained as a target product in the crude state.

Yield 57 mg Yield 137%

ESI-MS (NEG) m / z [Attribution]
Found: 568.9 Calc: 588.5 [(MH) ^- ]

Synthesis of KS9
Vacuum-dried 4 (57 mg, 96.9 μmol, FW588.5) was azeotroped twice with dehydrated pyridine (1.5 mL), suspended in dehydrated DMF (900 μL), and dehydrated Tributhyl amine (393.0 μmol, 94 μL, FW185 .35
, D = 0.775 g / mL), 0.5M Diphosphoric acid inDMF (485 μmol, 970 μL, FW177.98) was added and stirred at room temperature for 10 hours. After completion of the reaction, TEAB (3.7 mL) was added for quenching, and the mixture was concentrated by distillation under reduced pressure. The mixture was washed with diethyl ether, and the aqueous phase was distilled off under reduced pressure. It was dissolved in a small amount of distilled water and purified with an anion column. Further, purification was performed by HPLC purification, medium pressure column, and cation column.

Yield 232OD Yield 10.8%

ESI-MS (NEG) m / z [Attribution]
Found: 697.0 Calc: 698.4 [(MH) ^- ]

Selection (modified DNA) targeting Camptothecin derivative 1
<Buffer used in Selection>
Same as that used for screening of natural DNA aptamer.

<DNA used>
・ VIT # T1-H (5'-terminal HEX label * Modified DNA prepared using this as a template corresponds to Rnd '# 70. Used for gel affinity assay)
5'- CACCAGAGGCACGCCAGACANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCttCGCCGtGCtAGCCGtGt -3 'SEQ ID NO: 25
N represents A, G, C or modified T (compound 5 synthesized above).
・ VIT # P1-F (5 'terminal FAM labeling)
5′-CACCAGAGGCACGCCAGACA-3 ′ SEQ ID NO: 26
・ VIT # P2-H (5'-terminal HEX label)
5′-ACACGGCTAGCACGGCGAAG-3 ′ SEQ ID NO: 27
・ VIT # P1-P (5 'terminal phosphate labeling)
5′-CACCAGAGGCACGCCAGACA-3 ′ SEQ ID NO: 26
・ VIT # P1-N
5′-CACCAGAGGCACGCCAGACA-3 ′ SEQ ID NO: 26
・ VIT # P2-N
5′-ACACGGCTAGCACGGCGAAG-3 ′ SEQ ID NO: 27

<Preparation of affinity gel>
Same as screening of natural DNA aptamer.

<Synthesis of initial library> ^{* 2}
The initial library was prepared by a primer extension reaction using VIT # T1-H as a template strand and VIT # P1-F as a primer strand. Natural dATP, dCTP, dGTP and modified dTTP (5) were used for the substrate nucleoside triphosphate, and KOD Dash DNA polymerase was used for the polymerase. The single strand of the generated modified DNA can be separated and purified from the template strand VIT # T1-H by denaturing PAGE.

<Screening by Affinity Chromatography>
The modified DNA (110 pmol) obtained by PAGE purification was dissolved in 1 × LC-AT (400 μl) and refolded [94 ° C., 30 sec → 25 ° C. (0.5 ° C./min)] after UV measurement. The negative selection gel and the positive selection gel were substituted with Buffer 1 × LC-AT 400 μl × 4 times, and the DNA was incubated on the negative selection gel (37 ° C., 10 rpm, 1 h). The supernatant was collected, and after UV measurement, it was incubated on a gel for positive selection (37 ° C., 10 rpm, 1 h). The supernatant was collected and UV was measured. The positive selection gel was replaced with Buffer and unbound DNA was removed with 400 μl × 4 times of LC-A. (After 8 rounds, the buffer was replaced and unbound DNA was removed in 400 μl × 4 times.)
400 μL of elution buffer (LC-B for up to 7 rounds, LC-B2 for up to 8 rounds) was added to the gel for positive selection to elute DNA bound to the gel for positive selection (up to 3 rounds up to 3 rounds). Times, twice after the 8th round). The eluted solution was desalted with a spin column and freeze-dried, and then 162 μl of distilled water and 18 μl of 10 × KOD Dash Buffer were added to dissolve the DNA. PCR was performed using this solution.
The amount of DNA incubated on the gel for positive selection was measured by UV, and the amount of eluted DNA was measured by denaturing PAGE, and the progress of selection was confirmed from the ratio.

<Amplification of eluted DNA>
The modified DNA showing binding activity eluted at 37 ° C. was amplified by performing polymerase chain reaction (PCR). Take a small sample after the reaction, add 0.1% BPB solution containing 7M Urea 3mM EDTA, denature (94 ℃, 5min), and react by gel electrophoresis (200V, 35min, 45 ℃) using 10% denaturing PAGE Confirmed progress. In PCR, VIT # P1-P and VIT # P2-H are used as primers and KOD is used as an enzyme.
Dash DNA polymerase was used.

<Preparation of single-stranded template DNA>
Double-stranded DNA prepared by PCR was made into single strands by λexonuclease. Purified by denaturing PAGE as described above ^{* 1} .

<Preparation of single-stranded modified DNA>
Using the prepared single-stranded template strand and the primer strand VIT # P1-F, a primer extension reaction was performed in the same manner as described above ^{* 2} to prepare a single-stranded modified DNA. This was used for the next round of libraries.

<Aptamer isolation and sequence analysis>
Same as screening of natural DNA aptamer.

<Gel affinity assay>
Same as screening of natural DNA aptamer.

As a result of nine rounds of selection, the following sequences were obtained.
* T represents the modified base.

As a result of examining the binding of these sequences to camptothecins by Affinity Chromatography, the results are shown in Table 11.

In addition, the sequences in Table 12 were obtained. FIG. 1 shows the result of sequence analysis. Several consensus sequences have been discovered.

Claims (5)

A compound represented by the following formula (I) or a salt thereof:
A polynucleotide comprising a residue of a compound represented by the following formula (I):
The sequence of base numbers 21 to 50 of any of SEQ ID NOs: 28 to 32, 35 to 43 and 45 to 59, the sequence of base numbers 21 to 51 of SEQ ID NO: 44, or the sequence of base numbers 21 to 48 of SEQ ID NO: 60 A camptothecin binding agent comprising a nucleic acid aptamer containing (modified T in these sequences represents a residue of a compound represented by the following formula (I)).
The camptothecin binding agent according to claim 3, wherein the nucleic acid aptamer is single-stranded DNA. An anticancer agent comprising the camptothecin binding agent according to claim 3 and a camptothecin.