KR20220113132A - New compound for detecting pyrophosphate compound and method for preparing the same - Google Patents
New compound for detecting pyrophosphate compound and method for preparing the same Download PDFInfo
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- 235000011180 diphosphates Nutrition 0.000 title claims abstract description 35
- -1 pyrophosphate compound Chemical class 0.000 title claims description 14
- 238000000034 method Methods 0.000 title abstract description 9
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 claims abstract description 24
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 claims abstract description 19
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- 125000000524 functional group Chemical group 0.000 claims description 4
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- G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
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Abstract
Description
본 발명은 발색화합물과 하이드라존(hydrazone) 작용기를 이용한 구리이온(Cu2+) 착물의 개발 및 이를 이용한 파이로포스페이트(Pyrophosphate, ppi)를 감지하는 방법에 관한 것으로, 구체적으로 DNA 또는 RNA 증폭시에 나오는 파이로포스페이트 (Pyrophosphate)화합물을 감지할 수 있는 발색 및 형광을 나타내는 새로운 화합물을 개발한 것이다.The present invention relates to the development of a copper ion (Cu 2+ ) complex using a chromogenic compound and a hydrazone functional group and a method for detecting pyrophosphate (ppi) using the same, specifically, DNA or RNA amplification A new compound has been developed that exhibits color development and fluorescence that can detect pyrophosphate compounds from poetry.
전통적인 유전자 분자 진단 방법은 real-time PCR이 사용되며, real-time PCR 방법은 병원체의 유전자를 증폭하여 탐지함으로 극미량의 항원도 고민감도로 검출 가능하며 그 민감도와 보편성으로 인해 다양한 감염성 유전성 질병 및 미세한 유전자의 차이를 감별하기 위한 SNP Typing 에도 활발히 사용되고 있다.The traditional genetic molecular diagnostic method uses real-time PCR, and the real-time PCR method amplifies and detects pathogen genes so that even trace antigens can be detected with high sensitivity. It is also actively used for SNP Typing to discriminate genetic differences.
PCR(Polymerase Chain Reaction)을 포함한 DNA 복제 및 RNA 전사 등 다양한 유전자 증폭 과정 동안 필수적으로 수반하는 것이 파이로포스페이트(pyrophosphate)의 생성되는 것이다. 따라서 파이로포스페이트의 생성을 검출할 수 있다면 다양한 유전자의 증폭 정도를 확인할 수 있다.Generation of pyrophosphate is essential during various gene amplification processes such as DNA replication and RNA transcription including PCR (Polymerase Chain Reaction). Therefore, if the generation of pyrophosphate can be detected, the degree of amplification of various genes can be confirmed.
각종 유전자 분자 진단은 유전자 증폭을 이용하고 있으므로 파이로포스페이트(pyrophosphate)의 생성 검출은 다양한 유전자 분자 진단에 사용이 가능하다.Since the diagnosis of various gene molecules uses gene amplification, the detection of generation of pyrophosphate can be used for diagnosis of various gene molecules.
비색법을 이용한 파이로포스페이트의 생성 검출은 현장 유전자 분자 진단에 유용하며, 특별한 검출 기기의 사용 없이 발색의 변화 자체만으로 표적 유전자를 진단 할 수 있는 분자 진단 시스템은 앞으로 유전자 진단에서 효율적인 현장 진단 시스템으로 사용될 수 있다.The detection of the generation of pyrophosphate using a colorimetric method is useful for on-site genetic molecular diagnostics. can
현재까지 파이로포스페이트를 표적으로 비색법으로 검출하는 물질에 대해 개발이 미비하다.To date, development of a material for colorimetrically detecting pyrophosphate as a target is insufficient.
이에 본 발명자들은 파이로포스페이트를 비색법으로 검출할 수 있는 발색 화합물들을 개발하여 본 발명을 완성하였다. Accordingly, the present inventors completed the present invention by developing chromogenic compounds capable of detecting pyrophosphate by a colorimetric method.
따라서 본 발명은 1) 발색 화합물에 하이드라진을 반응시키는 단계; 2) 단계 1)의 생성물에 알데하이드(aldehyde) 작용기를 갖는 화합물 또는 케톤(keton) 작용기를 갖는 화합물과 반응시키는 단계; 및 3) 단계 2)의 생성물에 구리이온(Cu2+) 착물을 형성하는 단계에 의해 생성된 화합물을 포함하는 파이로포스페이트 화합물 감지용 조성물을 제공한다.Accordingly, the present invention comprises the steps of 1) reacting hydrazine with a chromogenic compound; 2) reacting the product of step 1) with a compound having an aldehyde functional group or a compound having a ketone functional group; And 3) provides a composition for detecting a pyrophosphate compound comprising a compound produced by the step of forming a copper ion (Cu 2+ ) complex in the product of step 2).
또한, 본 발명은 제 1항에 조성물을 포함하는 파이로포스페이트 화합물 감지용 키트를 제공한다.In addition, the present invention provides a kit for detecting a pyrophosphate compound comprising the composition of claim 1.
본 발명은 1) 발색 화합물에 하이드라진을 반응시키는 단계; 2) 단계 1)의 생성물에 알데하이드(aldehyde) 작용기를 갖는 화합물 또는 케톤(keton) 작용기를 갖는 화합물과 반응시키는 단계; 및 3) 단계 2)의 생성물에 구리이온(Cu2+) 착물을 형성하는 단계에 의해 생성된 화합물을 포함하는 파이로포스페이트 화합물 감지용 조성물을 제공한다.The present invention comprises the steps of 1) reacting hydrazine with a chromogenic compound; 2) reacting the product of step 1) with a compound having an aldehyde functional group or a compound having a ketone functional group; And 3) provides a composition for detecting a pyrophosphate compound comprising a compound produced by the step of forming a copper ion (Cu 2+ ) complex in the product of step 2).
본 발명에 있어서, 상기 발색 화합물은 인디고(Indigo), 나일 블루(Nile Blue), 메틸 블루(Methyl blue), 크리스털 바이올렛(Crystal violet), 메틸 레드(Methyl red), 메틸 그린(Methyl green) 및 로다민(rhodamine)으로 이루어진 군으로부터 선택되는 화합물인 것일 수 있다.In the present invention, the chromogenic compound is indigo, Nile blue, methyl blue, crystal violet, methyl red, methyl green, and It may be a compound selected from the group consisting of rhodamine.
본 발명에 있어서, 상기 단계 1)의 생성물은 하이드라진(hydrazine) 작용기를 포함하는 것일 수 있다.In the present invention, the product of step 1) may include a hydrazine functional group.
본 발명에 있어서, 상기 단계 1)의 생성물은 케톤(Ketone), 알데하이드(aldehyde), 카복실산(carboxylic acid), 아민(amine) 및 이민(imine)으로 이루어진 군으로부터 선택되는 작용기를 더 포함하는 것일 수 있다.In the present invention, the product of step 1) may further include a functional group selected from the group consisting of ketone, aldehyde, carboxylic acid, amine and imine. have.
본 발명에 있어서, 상기 알데하이드(aldehyde) 작용기를 갖는 화합물은 싸이오펜 알데하이드(thiophene aldehyde) 또는 Thiophene 2 carboxaldehyde, Pyridine 2 carboxaldehyde 4-hydoxy salicylaldehyde, Methyl thiosalicylate, 4 Diethylamino salicylaldehyde, 2-formyl thiazole 인 것일 수 있다.In the present invention, the compound having an aldehyde functional group may be thiophene aldehyde, Thiophene 2 carboxaldehyde, Pyridine 2 carboxaldehyde 4-hydoxy salicylaldehyde, Methyl thiosalicylate, 4 Diethylamino salicylaldehyde, or 2-formyl thiazole. .
본 발명에 있어서, 상기 단계 2)의 생성물은 하이드라존(hydrazone) 작용기를 포함하는 것일 수 있다.In the present invention, the product of step 2) may include a hydrazone functional group.
또한, 본 발명은 상기 조성물을 포함하는 파이로포스페이트 화합물 감지용 키트를 제공한다.In addition, the present invention provides a kit for detecting a pyrophosphate compound comprising the composition.
본 발명의 파이로포스페이트 화합물 감지용 화합물은 현장에서 질병관련 표적 유전자를 검출하는데 발색 신호를 나타내는 화합물들로 바로 적용이 가능하여 모든 유전자의 분자 진단하는데 유용하게 이용할 수 있다. The compound for detecting a pyrophosphate compound of the present invention can be directly applied as a compound showing a color development signal to detect a disease-related target gene in the field, and thus can be usefully used for molecular diagnosis of all genes.
도 1은 발색화합물과 구리이온(Cu2+)착물(E)를 이용한 파이로포스페이트(ppi) 비색 검출 방법에 관한 것이다:
가. Indigo-hydrazone-thiophene 구리이온(Cu2+)착물을 이용한 파이로포스페이트 (ppi) 비색 검출 방법,
나. Rhodamine-hydrazone-thiophene 구리이온(Cu2+) 착물을 이용한 파이로포스페이트(ppi) 비색 검출 방법.
도 2은 가능한 A 발색 화합물들을 나열한 그림이다;
인디고(Indigo), 나일 블루(Nile Blue), 메틸 블루(Methyl blue), 크리스털 바이올렛(Crystal violet), 메틸 레드(Methyl red), 메틸 그린(Methyl green) 및 로다민(rhodamine).1 relates to a method for colorimetric detection of pyrophosphate (ppi) using a chromogenic compound and a copper ion (Cu 2+ ) complex (E):
go. Pyrophosphate (ppi) colorimetric detection method using indigo-hydrazone-thiophene copper ion (Cu 2+ ) complex,
me. A method for colorimetric detection of pyrophosphate (ppi) using a rhodamine-hydrazone-thiophene copper ion (Cu 2+ ) complex.
Figure 2 is a diagram listing possible A chromogenic compounds;
Indigo, Nile Blue, Methyl blue, Crystal violet, Methyl red, Methyl green and rhodamine.
이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 1) 발색 화합물에 하이드라진을 반응시키는 단계; 2) 단계 1)의 생성물에 알데하이드(aldehyde) 작용기를 갖는 화합물 또는 케톤(keton) 작용기를 갖는 화합물과 반응시키는 단계; 및 3) 단계 2)의 생성물에 구리이온(Cu2+) 착물을 형성하는 단계에 의해 생성된 화합물을 포함하는 파이로포스페이트 화합물 감지용 조성물을 제공한다.The present invention comprises the steps of 1) reacting hydrazine with a chromogenic compound; 2) reacting the product of step 1) with a compound having an aldehyde functional group or a compound having a ketone functional group; And 3) provides a composition for detecting a pyrophosphate compound comprising a compound produced by the step of forming a copper ion (Cu 2+ ) complex in the product of step 2).
본 발명에 있어서, 상기 발색 화합물은 인디고(Indigo), 나일 블루(Nile Blue), 메틸 블루(Methyl blue), 크리스털 바이올렛(Crystal violet), 메틸 레드(Methyl red), 메틸 그린(Methyl green) 및 로다민(rhodamine)으로 이루어진 군으로부터 선택되는 화합물인 것일 수 있다.In the present invention, the chromogenic compound is indigo, Nile blue, methyl blue, crystal violet, methyl red, methyl green, and It may be a compound selected from the group consisting of rhodamine.
본 발명에 있어서, 상기 단계 1)의 생성물은 하이드라진(hydrazine) 작용기를 포함하는 것일 수 있다.In the present invention, the product of step 1) may include a hydrazine functional group.
본 발명에 있어서, 상기 단계 1)의 생성물은 케톤(Ketone), 알데하이드(aldehyde), 카복실산(carboxylic acid), 아민(amine) 및 이민(imine)으로 이루어진 군으로부터 선택되는 작용기를 더 포함하는 것일 수 있다.In the present invention, the product of step 1) may further include a functional group selected from the group consisting of ketone, aldehyde, carboxylic acid, amine and imine. have.
본 발명의 상기 과정은 하기 도 1에 구체적인 반응식으로 나타내었다.The process of the present invention is shown as a specific reaction scheme in FIG. 1 below.
케톤(Keton) 또는 알데히드(aldehyde)를 가진 발색화합물(Nile Blue, Methyl blue, Crystal violet, Methy red, Methyl green, Indigo 등) 도 1의 A에 하이드라존 작용기 B를 도입한다.A hydrazone functional group B is introduced into A of FIG.
하이드라존 작용기가 도입된 A 화합물에 알데하이드 또는 케톤 작용기를 가진 화합물 C와 반응하여 구리이온(Cu2+)와 선택적으로 착물을 만들 수 있는 하이드라존 작용기가 도입된 화합물을 합성한다. A compound having a hydrazone functional group introduced therein, which can selectively complex with copper ions (Cu 2+ ), is synthesized by reacting compound A with a hydrazone functional group with compound C having an aldehyde or ketone functional group.
이때 발색 화합물 A에 하이드라진 작용기가 도입될 수도 있고 반대로 연결될 화합물 C에 하이드라진 작용기가 도입되어 반응을 하여 A와 C가 하이드라존 작용기에 의해 연결된 생성물을 합성한다.At this time, a hydrazine functional group may be introduced into the chromogenic compound A, or a hydrazine functional group is introduced into the compound C to be linked to, and reacted to synthesize a product in which A and C are linked by a hydrazone functional group.
본 발명에 있어서, 상기 알데하이드(aldehyde) 작용기를 갖는 화합물은 싸이오펜 알데하이드(thiophene aldehyde) 또는 Thiophene 2 carboxaldehyde, Pyridine 2 carboxaldehyde 4-hydoxy salicylaldehyde, Methyl thiosalicylate, 4 Diethylamino salicylaldehyde, 2-formyl thiazole 것일 수 있다.In the present invention, the compound having the aldehyde functional group may be thiophene aldehyde, Thiophene 2 carboxaldehyde, Pyridine 2 carboxaldehyde 4-hydoxy salicylaldehyde, Methyl thiosalicylate, 4 Diethylamino salicylaldehyde, or 2-formyl thiazole.
본 발명에 있어서, 상기 단계 2)의 생성물은 하이드라존(hydrazone) 작용기를 포함하는 것일 수 있다.In the present invention, the product of step 2) may include a hydrazone functional group.
하이드라존 작용기에 의해 연결된 최종 발색 생성물에 구리이온(Cu2+)를 넣어 다양한 발색변화를 나타내는 발색 착물 E를 합성한다. 합성된 화합물은 순차적으로 구리이온(Cu2+)와 파이로포스페이트를 선택적으로 감지하는 발색화합물이어야 한다. 이 발색 착물은 비색을 가질 수도 있고 형광을 가질 수 도 있다.Copper ions (Cu 2+ ) are added to the final color product linked by hydrazone functional groups to synthesize color complex E showing various color changes. The synthesized compound should be a chromogenic compound that selectively detects copper ions (Cu 2+ ) and pyrophosphate sequentially. This chromogenic complex may have colorimetric or fluorescence.
파이로포스페이트를 민감하게 감지하여 발색변화가 있는 구리 발색 착물 E를 찾는다.Sensitively detect pyrophosphate to find copper color complex E with color change.
파이로포스페이트는 발색 착물의 구리이온(Cu2+)를 빼내어 파이로포스페이구리 착물을 형성한다.The pyrophosphate removes the copper ion (Cu 2+ ) of the chromogenic complex to form a pyrophosphate copper complex.
구리이온이 빠져나간 발색화합물은 비색의 변화 또는 형광의 변화를 유발한다(로다민의 경우 Lactam ring formation).The chromogenic compound from which copper ions escape causes a change in colorimetric or fluorescence (Lactam ring formation in the case of rhodamine).
이러한 발색 화합물은 MeCN, H2O, 또는 각종 buffer 용액에서 작동한다.These chromogenic compounds work in MeCN, H 2 O, or various buffer solutions.
이러한 발색화합물은 유전자(DNA, RNA)의 증폭과정에 기본적으로 수반되는 파이로포스페이트(pyrophosphate)를 색깔 변화로 탐지하는 물질로 사용될 수 있다.Such a chromogenic compound can be used as a material for detecting pyrophosphate, which is basically involved in the amplification process of genes (DNA, RNA), as a color change.
발색 착물은 구리이온(Cu2+)이외에 K+, Na+, Li+, Ca2+,Mg2+, Rb+, Co2+, Ni2+, Fe2+, Zn2+, Pb2+, Cu2+, Hg2+, Al3+, Ga3+, Fe3+, In3+를 포함할 수 있다. In addition to copper ions (Cu 2+ ), the color complex is K+, Na+, Li+, Ca 2+ , Mg 2+ , Rb+, Co 2+ , Ni 2+ , Fe 2+ , Zn 2+ , Pb 2+ , Cu 2+ , Hg 2+ , Al 3+ , Ga 3+ , Fe 3+ , and In 3+ .
이 발색 착물은 파이로포스페이트염(pyrophosphate, ppi)만 선택적으로 반응하여 색의 변화가 나타나며, adenosine monophosphate(AMP), adenosine diphosphate(ADP), dNTPs(dATP, dGTP, dCTP, dTTP), 그리고 I, Br, Cl, F,NO3, ClO4, H2PO4, SO42(Na+), HSO4, CO3 2, PO43,, AcO 에는 반응하지 않는다.This color complex selectively reacts with only pyrophosphate (ppi) to change color, and adenosine monophosphate (AMP), adenosine diphosphate (ADP), dNTPs (dATP, dGTP, dCTP, dTTP), and I, It does not react with Br, Cl, F,NO 3 , ClO 4 , H 2 PO 4 , SO4 2 (Na + ), HSO 4 , CO 3 2 , PO4 3 , , AcO.
이 발색 착물은 DTT(Dithiothreitol)와, 시스테인(cysteine), homocysteine (Hcyst), H2S, H2O2 와 반응하지 않는다.This chromogenic complex does not react with DTT (Dithiothreitol), cysteine, homocysteine (Hcyst), H 2 S, and H 2 O 2 .
이 발색 착물은 다양한 pH(3-11)에서 작동할 수 있다.This chromogenic complex can operate at various pHs (3-11).
또한, 본 발명은 상기 조성물을 포함하는 파이로포스페이트 화합물 감지용 키트를 제공한다.In addition, the present invention provides a kit for detecting a pyrophosphate compound comprising the composition.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다.Hereinafter, preferred examples are presented to help the understanding of the present invention.
그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 이에 의해 본 발명의 내용이 한정되는 것은 아니다.However, the following examples are only provided for easier understanding of the present invention, and thus the content of the present invention is not limited thereto.
<실시예 1> 발색화합물과 copper(Cu<Example 1> Coloring compound and copper (Cu) 2+2+ )착물(E)를 이용한 파이로포스페이트(ppi) 비색 검출 방법) Pyrophosphate (ppi) colorimetric detection method using complex (E)
화합물의 합성 단계는 크게 A, B, C, D, 및 E 파트로 나뉘어 설계되고 합성된다. 발색 화합물의 기본 구조는 기본적인 다양한 발색 화합물(A)에 하이드라진(hydrazine) 작용기를 도입하고 이를 또 다른 화합물 C와 반응시켜 하이드라존(hydrazone) 작용기를 포함한 D 화합물을 합성하고 이를 이용하여 구리이온(Cu2+)와 착물을 형성한 E 화합물을 합성한다(도 1).The synthesis step of the compound is largely divided into A, B, C, D, and E parts, designed and synthesized. The basic structure of the chromogenic compound is to synthesize a D compound including a hydrazone functional group by introducing a hydrazine functional group to the basic various chromogenic compound (A) and reacting it with another compound C, and using it to synthesize copper ion ( Cu 2+ ) and a complex E compound is synthesized (FIG. 1).
구리이온(Cu2+)와 착물을 형성한 발색화합물은 파이로포스페이트(pyrophosphate)를 만났을 때 구리와 파이로포스페이트 착물의 형성을 통해 자유로운 상태의 발색 화합물이 되며, 구리이온(Cu2+) 착물(E)에 파이로포스페이트(pyrophosphate)가 반응하면(F) 발색 화합물 마다 특이적인 색의 변화가 나타난다(도 1).When the chromogenic compound complexed with copper ions (Cu 2+ ) meets pyrophosphate, it becomes a chromogenic compound in a free state through the formation of a pyrophosphate complex with copper, and the copper ion (Cu 2+ ) complex When pyrophosphate reacts with (E) (F), a specific color change appears for each color-generating compound (FIG. 1).
A 부분은 인디고(Indigo), 나일 블루(Nile Blue), 메틸 블루(Methyl blue), 크리스털 바이올렛(Crystal violet), 메틸 레드(Methyl red), 메틸 그린(Methyl green) 및, 로다민(rhodamine) 등 다양한 발색화합물이 될 수 있다(도 2).Part A is Indigo, Nile Blue, Methyl blue, Crystal violet, Methyl red, Methyl green and, It may be a variety of chromogenic compounds, such as rhodamine (FIG. 2).
B 화합물은 기본적으로 하이드라진(hydrazine) 작용기를 포함하나 이외에 케톤(Ketone), 알데하이드(aldehyde), 카복실산(carboxylic acid), 아민(amine) 및 이민(imine)을 포함한 발색 화합물이다.Compound B basically includes a hydrazine functional group, but in addition to ketone, aldehyde, carboxylic acid, amine and imine are color-developing compounds including.
C 부분은 케톤(Ketone), 알데하이드(aldehyde), 카복실산(carboxylic acid), 아민(amine) 및 이민(imine) 등 다양한 작용기를 가진 싸이오펜 알데하이드(thiophene aldehyde) 또는 Thiophene 2 carboxaldehyde, Pyridine 2 carboxaldehyde 4-hydoxy salicylaldehyde, Methyl hiosalicylate, 4 Diethylamino salicylaldehyde, 2-formyl thiazole 등 전자가 풍부한 방향족(aromatic) 화합물들이 될 수 있다.Part C is thiophene aldehyde or Thiophene 2 carboxaldehyde, Pyridine 2 carboxaldehyde 4- with various functional groups such as ketone, aldehyde, carboxylic acid, amine and imine Electron-rich aromatic compounds such as hydoxy salicylaldehyde, methyl hiosalicylate, 4-diethylamino salicylaldehyde, and 2-formyl thiazole can be used.
D는 A 또는 B에 C를 반응하여 합성하며 하이드라존 작용기를 포함하여야 한다. D is synthesized by reacting C with A or B, and must include a hydrazone functional group.
E는 D에 구리이온(Cu2+, divalent copper)를 도입한 착물이다. 이 착물 E를 이용하여 파이로포스페이트(pyrophosphate) 물질을 검출한다.E is a complex in which copper ions (Cu 2+ , divalent copper) are introduced into D. A pyrophosphate substance is detected using this complex E.
Claims (7)
2) 단계 1)의 생성물에 알데하이드(aldehyde) 작용기를 갖는 화합물 또는 케톤(keton) 작용기를 갖는 화합물과 반응시키는 단계; 및
3) 단계 2)의 생성물에 구리이온(Cu2+) 착물을 형성하는 단계에 의해 생성된 화합물을 포함하는 파이로포스페이트 화합물 감지용 조성물.1) reacting hydrazine with a chromogenic compound;
2) reacting the product of step 1) with a compound having an aldehyde functional group or a compound having a ketone functional group; and
3) A composition for detecting a pyrophosphate compound comprising a compound produced by forming a copper ion (Cu 2+ ) complex in the product of step 2).
The kit for detecting a pyrophosphate compound comprising the composition of claim 1 .
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