KR101884455B1 - A triazole derivative as mass tag molecule capable of hydrogen isotope substitution for quantitative mass analysis and a method manufacturing the same - Google Patents

Abstract

The present invention relates to a triazole derivative as a hydrogen isotope substitutable mass tag molecule for quantitative mass spectrometry. The present invention relates to a triazole derivative as a mass tag molecule capable of hydrogen isotope substitution, a method for manufacturing the triazole derivative, and a method for using the derivative as a mass tag molecule. The method for manufacturing the triazole compound for mass tag comprises: reacting an azide compound with a compound containing a carbon triple bond under a copper catalyst to manufacture a triazole compound.

Description

[0001] The present invention relates to a triazole derivative as a mass labeling molecule capable of substituting hydrogen isotopes for quantitative mass analysis and a method for producing the triazole derivative,

The present invention relates to a triazole derivative as a mass tag molecule capable of hydrogen isotope substitution for quantitative mass spectrometry, and more particularly, to a triazole derivative as a mass tag molecule capable of hydrogen isotope substitution, And a method of using the derivative as a mass tag molecule.

Qualitative analysis such as molecular weight is possible, but quantitative analysis is difficult when mass spectrometry of trace organic matter. Quantitative comparison between proteins of normal cells and abnormal cells (eg cancer cells) in analysis of biologically important trace substances such as proteins and sugars is very important in the study of proteins.

In the case of abnormal cells, there are relatively more or fewer proteins compared to normal cells, and mass analysis can be used to analyze them. In this case, a mass tag substance capable of selectively binding to a specific amino acid of the protein is used. When such mass labeling substances include isotopes, the mass spectrometry such as NMR or mass spectroscopy can be used to perform relative quantitative analysis of the same proteins in normal and abnormal cells, using mass differences between isotopes.

In this connection, US Patent Publication No. 2008-0124807 discloses a protein mass label characterized by the use of a benzylamine derivative.

Korean Patent Laid-Open Publication No. 2012-0018674 discloses a method for quantitatively analyzing proteins and peptides by labeling chemical labels containing isotopes on a protein or peptide to be analyzed and then mass-analyzing the proteins or peptides.

Korean Patent Publication No. 2010-0009466 discloses a labeling agent having a dipeptide structure in which an N-terminal is acylated and a linker is attached to a C-terminus.

However, the technique disclosed in the above document has a problem that a compound used as a labeling substance is not stable at room temperature and accurate quantitative analysis can not be performed even if it is used as a labeling substance.

U.S. Published Patent Application No. 2008-0124807 Korea Patent Publication No. 2012-0018674 Korea Patent Publication No. 2010-0009466

It is an object of the present invention to provide a triazole derivative which can easily replace an isotope and stably maintain a substituted isotope and can be used as a mass labeling molecule for quantitative mass analysis.

It is another object of the present invention to provide a method for preparing triazole derivatives which can be quantitatively analyzed for proteins or sugar bodies by using a chelating agent stable at room temperature and substituting with isotopes of hydrogen at high temperatures as a labeling substance do.

To achieve these and other advantages and in accordance with the purpose of the present invention, as embodied and broadly described herein, And a carbon triple bond under a copper catalyst to produce a triazole derivative represented by the following formula (1): < EMI ID = 1.0 >

[Chemical Formula 1]

(Wherein R ₁ , R ₂ and R ₃ are independently hydrogen, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a cycloalkenyl group or an aryl group)

      In one embodiment of the present invention, the azide compound is 3-azido-1-propanol, and the compound containing the carbon triple bond is a carbazole derivative having a carbon triple bond.

      In one embodiment of the present invention, the azide compound is a dextrin derivative having an azide group, and the compound containing the carbon triple bond is a propargyl alcohol.

      In one embodiment of the present invention, the method further comprises the step of reacting the triazole derivative of Formula 1 at 60-120 ° C. to replace hydrogen with deuterium to produce a triazole derivative of Formula 2 .

(2)

(Wherein R ₁ , R ₂ and R ₃ are independently hydrogen, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a cycloalkenyl group or an aryl group)

      The present invention also provides a triazole derivative for mass labeling represented by the following formula (1), prepared by the above process.

[Chemical Formula 1]

(Wherein R ₁ , R ₂ and R ₃ are independently hydrogen, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a cycloalkenyl group or an aryl group)

The present invention also provides a triazole derivative for mass labeling represented by the following formula (2), which is produced by the above process.

(2)

(Wherein R ₁ , R ₂ and R ₃ are independently hydrogen, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a cycloalkenyl group or an aryl group)

The present invention also provides a mass labeling agent comprising the triazole derivative for mass labeling.

In addition, the present invention provides a method for producing a mass spectrometric composition, comprising the steps of: reacting the mass-labeled triazole derivative with a specific binding group of a protein or a saccharide; And a step of carrying out a quantitative analysis of a protein or a saccharide conjugated with a triazole derivative through mass spectrometry, for use in quantitative analysis of a protein or a saccharide.

The present invention can provide a triazole derivative which can easily displace isotopes and stably maintain the substituted isotopes and can be used as mass labeling molecules for quantitative mass spectrometry.

In addition, the present invention provides a method for preparing triazole derivatives which can be quantitatively analyzed for protein or sugar by using thionyl chloride as a labeling substance by substituting isotopes of hydrogen at a high temperature and kinetically stable at room temperature.

Figure 1 shows the click reaction of a compound containing an azide compound (RN ₃ ) and a carbon triple bond under a copper catalyst.
FIG. 2 shows the ¹ H NMR results of 3- [4- (9-phenyl-9H-carbazol-3-yl) -1H-1,2,3-triazol-1-yl] propan-1-ol.
FIG. 3 shows the ¹ H NMR results of 1- (3-hydroxypropyl) -3-methyl-4- (9-phenyl-9H-carbazol-3-yl) -1H-1,2,3-triazol-3-ium iodide .
Figure 4 shows ¹ H NMR analysis of 1- (3-hydroxypropyl) -3-methyl-4- (9-phenyl-9H-carbazol-3- yl) -1D-1,2,3-triazol- The results are shown.
FIG. 5 shows mass spectroscopy analysis of 1- (3-hydroxypropyl) -3-methyl-4- (9-phenyl-9H-carbazol-3-yl) -1D-1,2,3-triazol-3-ium iodide .
6 shows a method for synthesizing a triazole derivative from a dextrin derivative.
FIG. 7 shows the ¹ H NMR analysis results of Heptakis {6- (4-hydroxymethyl-1H- [1,2,3] triazol-1-yl) -6-deoxy} -β-cyclodextrin.
FIG. 8 shows the ¹ H NMR analysis results of Heptakis {6- (3-methyl-4-hydroxymethyl-1H- [1,2,3] triazolium) -6-deoxy} -β-cyclodextrin iodide.
9 shows the ¹ H NMR analysis results of Heptakis {6- (3-methyl-4-hydroxymethyl-1D- [1,2,3] triazolium) -6-deoxy} -β-cyclodextrin iodide.
FIG. 10 shows the ¹ H NMR analysis results of Heptakis {6- (3-methyl-4-hydroxymethyl-1H- [1,2,3] triazolium) -6-deoxy} -β-cyclodextrin iodide.
FIG. 11 is a graph showing the effect of heptakis {6- (3-methyl-4-hydroxymethyl-1H- [1,2,3] triazolium) -6-deoxy} -β-cyclodextrin iodide left to stand at room temperature for 1 week under D ₂ O solution ¹ H NMR analysis results.
FIG. 12 shows a mass spectroscopy analysis result of Heptakis {6- (3-methyl-4-hydroxymethyl-1H- [1,2,3] triazolium) -6-deoxy} -β-cyclodextrin iodide. FIG. 13 shows mass spectroscopy results of Heptakis {6- (3-methyl-4-hydroxymethyl-1D- [1,2,3] triazolium) -6-deoxy} -β-cyclodextrin iodide.
FIG. 14 is a graph showing the results of the reaction of 1- (3-hydroxypropyl) -3-methyl-4- (9-phenyl-9H-carbazol- mass spectroscopy of a mixture of 3-methyl-4- (9-phenyl-9H-carbazol-3-yl) -1D-1,2,3-triazol-3-ium iodide .
FIG. 15 is a graph showing the results of the reaction of 1- (3-hydroxypropyl) -3-methyl-4- (9-phenyl-9H-carbazol- mass spectroscopy of a mixture of 3-methyl-4- (9-phenyl-9H-carbazol-3-yl) -1D-1,2,3-triazol-3-ium iodide .

Hereinafter, the present invention will be described in detail based on examples. It is to be understood that the terminology, examples and the like used in the present invention are merely illustrative of the present invention in order to more clearly explain the present invention and to facilitate understanding of the ordinary artisan, and should not be construed as being limited thereto.

Technical terms and scientific terms used in the present invention mean what the person skilled in the art would normally understand unless otherwise defined.
In the present invention, it is understood that the triazole derivative of the formula (1) and the triazole derivative of the formula (2) have the structure of the formula (1) and the formula (2). That is, the triazole derivative of the formula (1) means a triazole derivative having the structure of the formula (1), and therefore the triazole derivative of the formula (1) means the triazole compound of the formula (1).

The present invention relates to an azide compound; And a carbon triple bond in the presence of a copper catalyst to produce a triazole derivative represented by the following formula (1).

[Chemical Formula 1]

(Wherein R ₁ , R ₂ and R ₃ are independently hydrogen, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a cycloalkenyl group or an aryl group)

The alkyl, alkenyl and alkynyl groups are preferably C1 to C20, and the cycloalkyl group and the cycloalkenyl group are preferably C3 to C30, The aryl group is preferably C6 to C50.

The alkyl group, the alkenyl group, the alkynyl group, the cycloalkyl group, the cycloalkenyl group and the aryl group may include a functional group such as a hydroxy, an amine, an amide, a halogen, an ester, an ether, a urethane, an azide, a ketone, an aldehyde, have.

The cycloalkyl group, the cycloalkenyl group and the aryl group may have a hetero atom in the ring and need not be composed of only a cycloalkyl group, a cycloalkenyl group or an aryl group, but may include a cycloalkyl group, a cycloalkenyl group or an aryl group.

,

등의 관능기를 포함할 수 있다. R ₁ , R ₂ and R ₃ can be used without limitation as long as they are capable of reacting with a specific binding group of a protein or a saccharide, and include maleimide, α-haloacetyl,

,

And the like.

The present invention is characterized by a triazole derivative which is capable of easily substituting an isotope and stably maintaining a substituted isotope. The hydrogen of the triazole derivative can be substituted for deuterium and used for mass labeling of protein or saccharide.

The triazole derivative of the present invention can be used as a mass labeling substance or a mass labeling reagent to accurately perform quantitative analysis of a protein or a sugar.

The triazole derivative can replace hydrogen with deuterium, which is an isotope of hydrogen, at a high temperature (60 to 120 ° C), and the deuterium substituted is stably maintained at room temperature, so that it can be stably analyzed in the analysis of proteins or saccharides.

Conversely, it is also possible to use deuterium again for quantitative analysis by replacing deuterium with hydrogen at a high temperature.

The triazole derivative is stable at a room temperature in a state of isotope substitution, that is, it is kinetically stable at room temperature, so that the triazole derivative remains unchanged and substituted with other isotopes. In order to substitute with another isotope, the substitution reaction may be carried out again at a high temperature.

      The azide compound can be used without limitation as long as it is a compound containing an azide group, and 3-azido-1-propanol and the like are preferable.

The compound containing the carbon triple bond may be used without limitation as long as it is a compound containing a carbon triple bond, and a carbazole derivative having a carbon triple bond is preferable.

As an example, the triazole derivative can be prepared through a click reaction of a compound having an azide compound (RN ₃ ) and a carbon triple bond under a copper catalyst through the following procedure. After the click reaction, an alkylation reaction can be performed to introduce cations (Fig. 1).

      Also, the azide compound may be a dextrin derivative having an azide group, and the compound containing the carbon triple bond may be propargyl alcohol (FIG. 6).

      The preparation method comprises reacting the triazole derivative of Formula 1 with microwave (60 to 120 ° C, 10 to 60 minutes, 10 to 60 W) to replace hydrogen with deuterium to prepare a triazole derivative of Formula 2 Step < / RTI >

(2)

(Wherein R ₁ , R ₂ and R ₃ are independently hydrogen, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a cycloalkenyl group or an aryl group)

The alkyl, alkenyl and alkynyl groups are preferably C1 to C20, and the cycloalkyl group and the cycloalkenyl group are preferably C3 to C30, The aryl group is preferably C6 to C50.

The alkyl group, the alkenyl group, the alkynyl group, the cycloalkyl group, the cycloalkenyl group and the aryl group may include a functional group such as a hydroxy, an amine, an amide, a halogen, an ester, an ether, a urethane, an azide, a ketone, an aldehyde, have.

The cycloalkyl group, the cycloalkenyl group and the aryl group may have a hetero atom in the ring and need not be composed of only a cycloalkyl group, a cycloalkenyl group or an aryl group, but may include a cycloalkyl group, a cycloalkenyl group or an aryl group.

,

등의 관능기를 포함할 수 있다. R ₁ , R ₂ and R ₃ can be used without limitation as long as they are capable of reacting with a specific binding group of a protein or a saccharide, and include maleimide, α-haloacetyl,

,

And the like.

The preparation method comprises stirring and filtering the triazole derivative prepared above; And purifying and drying.

      The present invention also relates to a triazole derivative for mass labeling represented by the following formula (1), which is produced by the above production method.

[Chemical Formula 1]

(Wherein R ₁ , R ₂ and R ₃ are independently hydrogen, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a cycloalkenyl group or an aryl group)

The alkyl, alkenyl and alkynyl groups are preferably C1 to C20, and the cycloalkyl group and the cycloalkenyl group are preferably C3 to C30, The aryl group is preferably C6 to C50.

The alkyl group, the alkenyl group, the alkynyl group, the cycloalkyl group, the cycloalkenyl group and the aryl group may include a functional group such as a hydroxy, an amine, an amide, a halogen, an ester, an ether, a urethane, an azide, a ketone, an aldehyde, have.

The cycloalkyl group, the cycloalkenyl group and the aryl group may have a hetero atom in the ring and need not be composed of only a cycloalkyl group, a cycloalkenyl group or an aryl group, but may include a cycloalkyl group, a cycloalkenyl group or an aryl group.

,

등의 관능기를 포함할 수 있다. R ₁ , R ₂ and R ₃ can be used without limitation as long as they are capable of reacting with a specific binding group of a protein or a saccharide, and include maleimide, α-haloacetyl,

,

And the like.

The present invention also relates to a triazole derivative for mass labeling represented by the following formula (2), which is produced by the above production method.

(2)

(Wherein R ₁ , R ₂ and R ₃ are independently hydrogen, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a cycloalkenyl group or an aryl group)

The alkyl, alkenyl and alkynyl groups are preferably C1 to C20, and the cycloalkyl group and the cycloalkenyl group are preferably C3 to C30, The aryl group is preferably C6 to C50.

The alkyl group, the alkenyl group, the alkynyl group, the cycloalkyl group, the cycloalkenyl group and the aryl group may include a functional group such as a hydroxy, an amine, an amide, a halogen, an ester, an ether, a urethane, an azide, a ketone, an aldehyde, have.

The cycloalkyl group, the cycloalkenyl group and the aryl group may have a hetero atom in the ring and need not be composed of only a cycloalkyl group, a cycloalkenyl group or an aryl group, but may include a cycloalkyl group, a cycloalkenyl group or an aryl group.

,

등의 관능기를 포함할 수 있다. R ₁ , R ₂ and R ₃ can be used without limitation as long as they are capable of reacting with a specific binding group of a protein or a saccharide, and include maleimide, α-haloacetyl,

,

And the like.

The final triazole derivative, 1- (3-hydroxypropyl) -3-methyl-4- (9-phenyl-9H-carbazol-3-yl) -1H-1,2,3-triazol-3-ium iodide And the structure synthesized by Heptakis {6- (3-methyl-4-hydroxymethyl-1H- [1,2,3] triazolium) -6-deoxy} -β-cyclodextrin iodide through NMR analysis.

The structure and stability of deuterium substituted triazole derivatives were confirmed by ¹ H NMR and ¹³ C NMR, and deuterium substitution was identified using a MicroQ-TOF III (ESI) mass spectrometer .

The triazole derivative of the present invention was easily replaced with deuterium at a high temperature, and the deuterium substituted structure was stably maintained at room temperature.

       The deuterium-substituted triazole derivative of the present invention can be used as a mass marker to quantitatively analyze proteins and saccharides by binding thereto, and to confirm the structure and binding of proteins and saccharides at room temperature.

The present invention also relates to a mass labeling molecule, a mass labeling substance or a mass labeling agent comprising the triazole derivative for mass labeling.

The triazole derivative can selectively label proteins and saccharides by reacting with specific binding groups of proteins and saccharides.

In addition, the present invention provides a method for producing a mass spectrometric composition, comprising the steps of: reacting the mass-labeled triazole derivative with a specific binding group of a protein or a saccharide; And a step of performing quantitative analysis of a protein or a saccharide conjugated with a triazole derivative through mass spectrometry. The present invention relates to a method for quantitatively analyzing a triazole derivative for mass labeling.

Hereinafter, the present invention will be described in detail with reference to examples. The following examples are intended to illustrate the practice of the present invention and are not intended to limit the scope of the present invention.

(Example 1)

Preparation of 3- [4- (9-phenyl-9H-carbazol-3-yl) -1H-1,2,3-triazol-1-yl] propan-

3-ethynyl-9-phenyl-9H-carbazole (0.9 g) was dissolved in DMF (8 ml). (0.37 g, 3.72 mmol, 2 eq.), DIPEA (0.24 g, 1.86 mmol, 1 eq.) And CuI (30 mg) , And cuprisorb (2 g) was added thereto, stirred for one day, and filtered.

The filtered material was purified by silica column chromatography (eluent: n-hexane / ethyl acetate) and dried under vacuum to obtain 3- [4- (9-phenyl-9H-carbazol- -1,2,3-triazol-1-yl] propan-1-ol in 95% yield.

^One≪ 1 > H NMR (600 MHz, CDCl3₃): 隆 8.64 (d, 1H), 8.18-8.16 (d, 1H), 7.89 (s, 1H), 7.85-7.83 (q, 1H), 7.62-7.59 (q, 2H), 7.56-7.54 2H), 7.49-7.46 (m, 1H), 7.42-7.39 (q, 3H), 7.30-7.28 -2.19 (m, 2 H);

¹³≪ 1 > C NMR (150 MHz, CDCl3₃):? 141.3, 140.7, 137.4, 129.9, 129.8, 127.6, 127.0, 126.9, 126.2, 124.0, 123.7, 123.3, 122.5, 120.5, 120.2, 119.6, 117.7, 110.1, 109.9, 58.8, 47.0,

(Example 2)

Preparation of 1- (3-hydroxypropyl) -3-methyl-4- (9-phenyl-9H-carbazol-3- yl) -1H-1,2,3-triazol-3-ium iodide

3-yl) -1H-1,2,3-triazol-1-yl] propan-1-ol (0.1 g, 0.27 mmol) was added to 5 ml of DMF Melted. The melted solution was added _{CH 3 I (0.077g, 0.4mmol,} 2 eq) and allowed to react at 70 ℃ for 46 hours, then filtered to remove the solvent.

The filtered material was purified by silica column chromatography (developing solvent: DCM / MeOH) and dried under vacuum to give 1- (3-hydroxypropyl) -3-methyl-4- (9- 3-yl) -1H-1,2,3-triazol-3-ium iodide in a yield of 60%.

^One(M, 3H), 7.66-7.65 (d, 2H), 7.66-7.67 (d, IH) (T, 2H), 4.37 (s, 2H), 7.61-7.57 (q, 2H), 7.53-7.51 (d, 1H), 7.42-7.39 3H), 3.56-3.53 (q, 2H), 2.17-2.15 (t, 2H)

FIG. 2 relates to ¹ H NMR of the synthesized 3- [4- (9-phenyl-9H-carbazol-3-yl) -1H-1,2,3-triazol-1-yl] propan- ,

Figure 3 shows the ¹ H NMR analysis of 1- (3-hydroxypropyl) -3-methyl-4- (9-phenyl-9H-carbazol-3- yl) -1H-1,2,3-triazol-3-ium iodide Results are shown.

(Example 3)

Preparation of 1- (3-hydroxypropyl) -3-methyl-4- (9-phenyl-9H-carbazol-3-yl) -1D-1,2,3-triazol-3-ium iodide

3-yl) -1,3-triazol-3-ium iodide (10 mg) was added to a CD ₃ OD (1 ml) and reacted using microwave (65 ° C, 30 minutes, 50W).

^OneAs a result of 1 H NMR analysis, it was confirmed that the substitution of deuterium disappeared the peak of hydrogen from the triazole (Fig. 4).

^One≪ 1 > H NMR (600 MHz, CD₃(M, 4H), 7.52-7.49 (t, 1 H), 7.41-7.39 (m, 4H), 7.60-7. (d, IH), 7.37-7.36 (t, 2H), 4.57 (s, IH), 4.39 (d, 3H), 3.75-3.73 (t, 2H), 2.33-3.31 (t, 2H);

GC-MS (FAB, positive): calculated 384.19 for C ₂₄ DH ₂₂ N ₄ O ⁺ , observed 384 for [M] +

(Example 4)

Stability test at room temperature of 1- (3-hydroxypropyl) -3-methyl-4- (9-phenyl-9H-carbazol-3-yl) -1D-1,2,3-triazol-3-ium iodide

To test the stability of 1- (3-hydroxypropyl) -3-methyl-4- (9-phenyl-9H-carbazol-3-yl) -1D-1,2,3-triazol-3-ium iodide at room temperature The powder thus formed was allowed to stand at 25 ° C. for 1 week, and the structural change was measured by mass spectroscopy (FIG. 5).

The mass spectroscopy of the resulting powder shows no change with time, so that it is confirmed that the deuterium substituted triazole derivative is stable at room temperature.

(Example 5)

Preparation of Heptakis {6- (4-hydroxymethyl-1H- [1,2,3] triazol-1-yl) -6-deoxy} -β-cyclodextrin

Heptakis-6- (deoxy-6-azido) -β-cyclodextrin (1 g, 0.76 mmol) was dissolved in 4 ml of DMF. (0.46 mL, 7.98 mmol, 1.5 eq.), DIPEA (0.98 mL, 5.32 mmol, 1 eq.) And CuI (0.03 g, 0.16 mmol) , Cuprisorb (1 g) was added, and the mixture was stirred for one day to remove copper and then filtered.

Evaporate through rotary evaporator to remove DMF and propargyl alcohol and sonicate with cold methanol (35 mL) to remove DIPEA. After sonication, the precipitate was filtered under pressure and dried under vacuum to give 67.1% of heptakis {6- (4-hydroxymethyl-1H- [1,2,3] triazol-1-yl) -6-deoxy} ≪ / RTI >

^One≪ 1 > H NMR (600 MHz, DMSO-d₆): [delta] 7.81 (s, 1H, H₇1H), 5.97-5.96 (d, 1H, OH-2), 5.82_One), 4.35-4.30 (q, 3 H, H₉, H₆), 4.26-4.22 (q, 2H, H₅, H₆), 3.98 (s, 1H, H₃), 3.66-3.64 (d, 1H, H₂), 3.28-3.27 (d, 1H, H₄)

(Example 6)

Preparation of Heptakis {6- (3-methyl-4-hydroxymethyl-1H- [1,2,3] triazolium) -6-deoxy} -β-cyclodextrin iodide

Heptakis (1 g, 0.587 mmol, 1 eq.) Was dissolved in DMF (4 ml), CH ₃ I ( 511.8 [mu] L, 8.22 mmol, 14 equivalents). The mixture was stirred at 60 ° C under a nitrogen atmosphere and the reaction was confirmed by TLC (2-propanol: ethyl acetate: water: 28% ammonia = 6: 1: 3: 1). The reaction mixture was evaporated on a rotary evaporator to remove DMF, ). After solid precipitation, the precipitate was filtered under pressure, washed with methanol and then ether, and dried under vacuum

Heptakis {6- (3-methyl-4-hydroxymethyl-1H- [1,2,3] triazolium) -6-deoxy} -β-cyclodextrin iodide in a yield of 81.5%.

^One≪ 1 > H NMR (600 MHz, D₂O): [delta] 8.45 (s, 1H, H₇), 5.07-5.06 (d, 1H, H_One), 5.00-4.98 (t, 1H, H₆), 4.84-4.81 (q, 1H, H₆), 4.72-4.70 (d, 2H, H₉), 4.57-4.56 (m, 1H, H₅), 4.24-4.15 (t, 3H, CH₃), 3.94-3.91 (t, 1H, H₃), 3.43-3.41 (q, 1H, H₂), 3.31-3.28 (t, 1H, H₄)

¹³C NMR (214 MHz, D₂O): 38.54 (CH₃), 52.33 (C 9), 53.89 (C 6), 68.57 (C 5), 71.37 (C 2), 72.01 (C 3), 81.14 (C 4), 101.75 (C 1), 130.97

ESI-MS: m / z 1221 [M-2I] 2+, 772 [M-3I] 3+, 258 [M-7I] 7+

Figure 7

Heptakis {6- (4-hydroxymethyl- 1H- [1,2,3] triazol-1-yl) -6-deoxy} relates to ¹ H NMR analysis of the -β-cyclodextrin,

Figure 8

Heptakis {6- (3-methyl- 4-hydroxymethyl-1H- [1,2,3] triazolium) -6-deoxy} relates to ¹ H NMR analysis of the -β-cyclodextrin iodide.

(Example 7)

Preparation of Heptakis {6- (3-methyl-4-hydroxymethyl-1D- [1,2,3] triazolium) -6-deoxy} -β-cyclodextrin iodide

Heptakis {6- (3-methyl-4-hydroxymethyl-1H- [1,2,3] triazolium) -6-deoxy} -β-cyclodextrin iodide (0.1 g, 0.037 mmol) was added to D ₂ O microwave (100 캜, 30 min, 30 W).

^OneAs a result of 1 H NMR analysis, it was confirmed that the substitution of deuterium disappeared (H7 peak) from the hydrogen of the triazole (FIG. 9).

^One≪ 1 > H NMR (600 MHz, D₂O): [delta] 5.15 (s, 1H, H_One), 5.08-5.05 (d, 1H, H₆), 4.90-4.88 (d, 1H, H₆), 4.81-4.80 (d, 2H, H₉), 4.26-4.23 (t, 3H, CH₃), 4.02-3.98 (m, 1H, H₃), 3.53-3.52 (d, 1H, H₂), 3.41-3.38 (t, 1H, H₄)

¹³≪ 1 > C NMR (150 MHz, D₂O): 38.55 (CH₃), 52.30 (C 9), 53.82 (C 6), 68.50 (C 5), 71.36 (C 2), 71.99 (C 3), 81.12 (C 4), 101.73 (C 1), 130.67

ESI-MS: m / z 1224 [M-2I] 2+, 774 [M-3I] 3+, 259 [M-7I] 7

(Example 8)

Stability test of Heptakis {6- (3-methyl-4-hydroxymethyl-1H- [1,2,3] triazolium) -6-deoxy} -β-cyclodextrin iodide at room temperature

To test the stability of Heptakis {6- (3-methyl-4-hydroxymethyl-1H- [1,2,3] triazolium) -6-deoxy} -β-cyclodextrin iodide at room temperature, ₂ O solution, and ¹ H NMR peak change was observed (Table 1, Figs. 10 and 11).

It can be confirmed that the hydrogen-substituted triazole derivative is stable at room temperature since the relative integral ratio of H7 of triazole and H3 in cyclodextrin, which can be substituted with double hydrogen, hardly changes with time.

H7 integral value H3 integral value Antagonistic
(H7: H3) Early 42.97 57.13 0.75: 1 After 1 week at 25 ° C 44.80 55.20 0.81: 1

12 is a cross-

Mass spectroscopy of Heptakis {6- (3-methyl-4-hydroxymethyl-IH- [1,2,3] triazolium) -6-deoxy} -? - cyclodextrin iodide,

Figure 13

Mass spectroscopy of Heptakis {6- (3-methyl-4-hydroxymethyl-1D- [1,2,3] triazolium) -6-deoxy} -β-cyclodextrin iodide.

Since the hydrogen-substituted triazole derivative and the deuterium-substituted triazole derivative have different peaks from each other, the triazole derivative can be effectively used for quantitative analysis of protein or sugar.

FIG. 14 is a graph showing the results of the reaction of 1- (3-hydroxypropyl) -3-methyl-4- (9-phenyl-9H-carbazol- mass spectroscopy of a mixture of 3-methyl-4- (9-phenyl-9H-carbazol-3-yl) -1D-1,2,3-triazol-3-ium iodide .

FIG. 15 is a graph showing the results of the reaction of 1- (3-hydroxypropyl) -3-methyl-4- (9-phenyl-9H-carbazol- mass spectroscopy of a mixture of 3-methyl-4- (9-phenyl-9H-carbazol-3-yl) -1D-1,2,3-triazol-3-ium iodide .

2: 7 (H: D) 7: 2 (H: D) 383 m / z
peak intensity 109.26 1,325.51 384 m / z
peak intensity 350.00 390.73 peak intensity ratio 2: 7 (H: D) 7: 2 (H: D)

The hydrogen-substituted triazole derivative and the double-hydrogen substituted triazole derivative were mixed at an arbitrary mass ratio and the GC-MS was measured. The ratio of the peak intensities of 383m / z and 384m / z is the same as the mass ratio of the mixed materials. From these results, it is confirmed that quantitative analysis is possible.

Claims (8)

Azide compounds; And a carbon triple bond under a copper catalyst to prepare a triazole compound represented by the following formula (3) or a triazole compound represented by the following formula (4).

(3)

[Chemical Formula 4]

The method according to claim 1,
Wherein the triazole compound of Formula 3 is reacted at 60 to 120 占 폚 to prepare a triazole compound of Formula 5 by replacing hydrogen with deuterium.

[Chemical Formula 5]

The method according to claim 1,
Wherein the triazole compound of Formula 4 is reacted at 60 to 120 캜 to replace hydrogen with deuterium to produce a triazole compound of Formula 6 below.

[Chemical Formula 6]

The triazole compound for mass labeling according to claim 1,
Wherein the triazole compound for mass labeling is a triazole compound represented by the following formula (3) or a triazole compound represented by the following formula (4).

(3)

[Chemical Formula 4]

A triazole compound for mass labeling represented by the following formula (5), which is produced by the production method of claim 2.

[Chemical Formula 5]

A triazole compound for mass labeling according to claim 6, which is produced by the process of claim 3.

[Chemical Formula 6]

A mass labeling agent comprising the triazole compound for mass labeling according to any one of claims 4 to 6.
Reacting the mass labeling triazole compound of any one of claims 4 to 6 with a specific binding group of a protein or a sugar; And
Wherein the triazole compound for mass labeling is used for quantitative analysis of a protein or a saccharide, comprising the step of quantitatively analyzing the protein or saccharide conjugated with the triazole compound through mass spectrometry.

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