WO2015084075A1 - 쿠커비투릴-당 복합체 및 이의 제조방법 - Google Patents

쿠커비투릴-당 복합체 및 이의 제조방법 Download PDF

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WO2015084075A1
WO2015084075A1 PCT/KR2014/011834 KR2014011834W WO2015084075A1 WO 2015084075 A1 WO2015084075 A1 WO 2015084075A1 KR 2014011834 W KR2014011834 W KR 2014011834W WO 2015084075 A1 WO2015084075 A1 WO 2015084075A1
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sugar
cucurbituril
complex
formula
present
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English (en)
French (fr)
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김기문
장윤정
나타라잔 라말링감
고영호
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Institute for Basic Science
POSTECH Academy Industry Foundation
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Institute for Basic Science
POSTECH Academy Industry Foundation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains four or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H99/00—Subject matter not provided for in other groups of this subclass

Definitions

  • the present invention relates to a cooker bituril-sugar complex and a method for producing the same.
  • Artificial saccharide receptors can be used for applications such as sensing and drug delivery. Artificial saccharide receptors, which act on the basis of non-covalent bonds in aqueous solutions, have emerged as very interesting topics in biochemistry or supramolecular chemistry. have.
  • saccharide receptors the first and the most difficult is the separation of sugars and water molecules and the subsequent association of selective saccharides.
  • saccharide phosphoproteins There is only a handful of artificial waters with functional groups capable of hydrogen bonding with hydrophobic cages, such as lectins.
  • Aminosaccharides on the surface of the cell, are mostly present in the form of N-acylated compounds, which serve as biomarkers for many biological processes and diseases. They are also known as aminoglycosides. It is also an important factor.
  • boric acid-based receptors exist, but they have very weak bonds and metal ions are also recognized as aminosaccharides, but their mating is limited to detection, so that they are based on non-covalent bonds in aqueous solutions. Research is highly required.
  • the present invention detects artificial lectins for glycan analysis and detects cell-cell interactions.
  • Lipid-CB [7] Cookerbituril useful for the transport and detection of conjugates and antibody drugs. Provided are the complexes and methods for their preparation.
  • the present invention provides a cooker bituryl-sugar complex formed by supramolecular chain cooker bituril which recognizes and supports saccharides in an aqueous solution.
  • the cooker bituril according to an embodiment of the present invention may be represented by Chemical Formula 1.
  • X is 0, S or NH
  • A, and ⁇ 2 are each independently selected from H, OR, SR, NHR, COOH, 0 (CH 2) a R or 0 (CH 2) a SR, wherein a is 1 to 5 can Protocol, the R is H, halogen, C1-C30 alkyl, C2-C30 alkenyl, C2-C30 alkynyl, C2-C30 carbonylalkyl, C3-C30 cycloalkyl, C2-C30 heterocycloalkyl, C6-C30 aryl, C6- C30 aryl CI-C30 alkyl ,
  • n is an integer between 4 and 14.
  • X is 0, A, and A 2 are independently H, OR, 0 (CH 2 ) aR or 0 (CH 2 ) aSR, A is an integer of 1 to 5, and R is H, C1-C30 alkyl or C2-C30 alkenyl; n can be an integer from 6 to 10.
  • the sugar may be a monosaccharide, a disaccharide, or a polysaccharide, and preferably a sugar having an amino group or a salt containing the same.
  • a sugar having an amino group or a salt containing the same may be represented by an amino sugar antibiotic or the following formula (2).
  • R n to R l5 are independently of each other OH, NH 2 or NH 3 + X—, and any one of R n to R l5 is necessarily NH 2 or NH 3 + X-; [27] X- is a monovalent ion of acid.]
  • the cooker bituril fructose according to one embodiment of the present invention may have an equivalent ratio of 0.5 to 3: 1.
  • the present invention also provides a method for producing a cucurbituryl-sugar complex in which the cucurbituryl sugar is selectively recognized, supported and stabilized in an aqueous solution.
  • a cooker bituril of the present invention is prepared according to one embodiment of the method for preparing a sugar complex.
  • the cucurbituril may be represented by Chemical Formula 1, and in Chemical Formula 1, X is 0, A, and ⁇ are each independently H, OR, 0 (CH 2 ) aR or 0 (CH 2 ) aSR, a is an integer of 1 to 5, wherein R is H, C1-C30 alkyl or
  • n can be an integer from 6 to 10.
  • It may be a sugar having an amino group or a salt containing the same, and specifically, may be represented by an amino sugar antibiotic or the above Chemical Formula 2.
  • Cooker beryl fructose can have an equivalent ratio of 0.5 to 3: 1.
  • the present invention also provides a method for detecting sugars using cooker bituril. Effects of the Invention
  • the cooker bituril of the present invention-the sugar complex of the present invention in which the cooker bituril selectively recognizes sugars in the aqueous solution, which is supported by the cooker bituril joint and supported by the supported sugars and anomers.
  • cooker bituril By stabilizing by preventing the rotation of the light, cooker bituril can support and stabilize specific anomers to form an optional cooker bituryl-sugar complex according to the type of sugar.
  • the cucurbituril-glycocomplex of the present invention also introduces functionalization of cucurbituril outward rings to allow the analysis of artificial lectins or cells and cells for glycan analysis.
  • the cucurbituril-sugar complex of the present invention can also be used for the transport and detection of specific antibody drugs.
  • cooker bituril of the present invention cooker bituril can recognize sugars in aqueous solution and can detect cell-cell interactions.
  • the production method of the cooker bituril-sugar complex of the present invention can be prepared in aqueous solution.
  • Cooker bituril can recognize sugars and make stabilized cooker bituryl-sugar complexes by co-operating with cooker bituril joint, and can be applied to cell-cell interactions and transport and detection of antibody by the method of producing cooker bituryl-sugar complex of the present invention. have. Brief description of the drawings
  • 1 is a diagram showing the structure of a cooker bite reel
  • FIG. 4 shows CB [7]. This diagram shows the NMR spectrum of one complex.
  • Fig. 4 is a diagram showing the MALDI-TOF mass spectrum of CB [7] ⁇ one composite, [44] Fig. 5 is CB [7]. 1 complex and 1 (bottom) proton-coupled 13 C-NMR spectrum
  • FIG. 6 shows the ROESY (278K) of CB [7] .1 complex formed from two equivalents of CB].
  • FIG. 7 is a diagram showing an NMR spectrum of a CB [7] -2 complex.
  • Fig. 8 is a diagram showing the MALDI-TOF mass spectrum of CB [7] -two complexes.
  • Fig. 9 is CB [7]. 2 complex and 2 (bottom) proton-coupled 13 C-NMR spectrum
  • FIG. 10 is a diagram showing a DQF-COSY spectrum of a CB [7] .complex formed from two equivalents of CB [7].
  • Figure 11 shows the ROESY (278K) of CB [7] .2 complex formed from two equivalents of CB [group].
  • Figure 12 is a molecular dynamics modeling of B [7] -two complexes
  • FIG. 13 is a diagram showing an NMR spectrum of 2 when a CB group is added.
  • 15 is a diagram showing a 1 H NMR of a CB [7] .3 complex formed from two equivalents of CB [group].
  • FIG. 16 shows CB [3 and 10 (mM) ITC profiles in water at 298 K
  • FIG. 17 shows the ⁇ -NMR spectrum of 4 when CB [added]
  • FIG. 18 shows a CB [4] and 4 (10 mM) ITC profile in water at 298K.
  • the present invention was completed by preparing a cucurbituril-sugar complex.
  • the artificial acceptor is an ultra-molecular chain cucurbituril (CB [n]).
  • the present invention was found to selectively recognize saccharides in aqueous solution, and to stabilize the sugars with alpha or beta-omers by preventing the photochromic phenomena of sugars supported on agacoucurbituril.
  • Cookerbitu [n] reel (CB [n]) is a large ring molecule with a hard, hydrophobic cavity inside, which is a supermolecular molecule that dissolves in water without charge and is present in the cavity and inlet of CB [n].
  • Carbonyl groups enable stable formation of the master-guest complex based on hydrophobic interactions and strong ion-dipole interactions, but no stable cucurbituryl-sugar complexes supported on cucurbituryl sugars have been reported.
  • the cooker bituril of the present invention cooker bituril can be applied to transport and detect aminoglucoside antibody drugs by recognizing and stabilizing sugars in aqueous solution and by co-curing with the cooker bituril joint.
  • the cooker bituril of the present invention can recognize sugars.
  • a functional group can be introduced into the cucurbituril outbound ring to prepare an artificial lectin for glycan analysis or a lipid-CB [7] conjugate for detecting cell-cell interactions.
  • Cooker bituril according to an embodiment of the present invention may be represented by the following formula (1) 68 [68]
  • X is O, S or NH
  • A, and A 2 are each independently H, OR, SR, NHR, COOH, 0 (CH 2 ) a R or 0 (CH 2 ) a SR, wherein a is an integer of 1 to 5, R is H, halogen, C1-C30 alkyl, C2-C30 alkenyl, C2-C30 alkynyl, C2-C30 carbonylalkyl, C3-C30 cycloalkyl, C2-C30 heterocycloalkyl, C6-C30 aryl, C6- C30 aryl C1-C30 alkyl,
  • n is an integer between 4 and 14.
  • X is O
  • a 2 are independently H, OR, 0 (CH 2 ) aR or 0 (CH 2 ) aSR, Where a is 1 to 5 An integer, wherein R is H, C 1 -C 30 alkyl or C 2 -C 30 alkenyl; n could be an integer from 6 to 10.
  • X is 0, and ⁇ is independently H, OR, and R may be H, C1-C30 alkyl.
  • Substituents containing an "alkyl” moiety include both straight and branched forms.
  • aryl described in the present invention is aromatic by one hydrogen removal.
  • Organic radicals derived from hydrocarbons each comprising a single or fused ring system containing 4 to 7, preferably 5 or 6 ring atoms, each of which has a plurality of aryls connected in a single bond.
  • polycyclic heteroaryls condensed with one or more benzene rings which may be partially saturated.
  • Heteroaryls in the present invention also include forms in which one or more heteroaryls are linked by a single bond.
  • cycloalkyl described in the present invention means a saturated hydrocarbon ring
  • it may be a 5 to 7 membered alicyclic ring, including when the aromatic or alicyclic ring is fused.
  • Heterocycloalkyl described in the present invention . Is a heteroatom containing an oxygen, sulfur or nitrogen in the ring, or a cyclic ring, the number of hetero atoms is 1-4, preferably 1-2.
  • cycloalkyl is preferably monocyclo Aryl or alkyl or bicycloalkyl, which is an aromatic ring
  • C2-C30 carbonylalkyl, C3-C30 cycloalkyl and C2-C30 heterocycloalkyl are preferably C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C2-C10 carbonylalkyl, C3-C10 cyclo Alkyl and C2-C10 heterocycloalkyl, and C6-C30 aryl and C4-C30 heteroaryl can be C6-C12 aryl and C4-C12 heteroaryl.
  • Examples include monosaccharides to polysaccharides, include sugar antibiotics, preferably sugar antibiotics, monosaccharides, disaccharides or polysaccharides, preferably sugars with amino groups or salts containing them, or
  • It may be an aminosugar antibiotic, and more preferably an aminosugar antibiotic having an amino group or a salt containing the monosaccharide and an amino group.
  • Sugar antibiotics and sugar antibiotics of the present invention include sugars based on antibiotics and Antibiotics include antibiotics with or without functional groups and aminosugar antibiotics.
  • the aminosugar antibiotics of the present invention are based on sugars having amino groups.
  • Antibiotics include, for example, neomycin and ferromycin.
  • the sugars and amino sugar antibiotics having an amino group or a salt containing the same include, for example, D-glucosamine hydrochloride, D-galactosamine hydrochloride, emannosamine hydrochloride, and 6 -amino ⁇ 6.
  • D-glucosamine hydrochloride D-galactosamine hydrochloride
  • emannosamine hydrochloride emannosamine hydrochloride
  • 6 -amino ⁇ 6 -amino ⁇ 6.
  • -Dioxy-D-glucose, N-acetylglucosamine, sialic acid, L-daunosamine), neomycin and ferromycin but are not limited to these.
  • a sugar having an amino group or a salt containing the same may be represented by the following Chemical Formula 2.
  • R u to R l5 are each independently OH, NH 2 or NH 3 + X—, and any one of R n to R l5 is NH 2 or NH 3 + X-;
  • X- is a monovalent ion of acid.
  • R "to R 15 are independently a OH or NH 3 + X- with each other, to R l5 is a slow Chinese Language It must be NH 3 + X-.
  • X- can be any monoanion of an acid, for example, hydrochloric acid (C1), silver nitrate (N0 3- ), acetate ions (CH 3 COO). And preferably hydrochloric acid.
  • the cucurbit reel fructose according to one embodiment of the present invention has a molar ratio of 0.5 to 3: 1 days.
  • the present invention also includes a method for producing a cucurbituryl-sugar complex comprising contacting the cucurbituril and a sugar in an aqueous solution to produce a cucurbituryl-sugar complex.
  • the production of the cooker bituril-sugar complex of the present invention may melt the cooker bituryl fructose in an aqueous solution and contact the cooker bituril fructose, and the mixture of cooker bituryl fructose may be added to the aqueous solution so as to contact the cooker bituril fructose in the aqueous solution. Regardless of whether it is possible.
  • Cooked beryl fructose can have an equivalent ratio of 0.5 to 3: 1.
  • reaction time for adding the sugars of step (b) to make the cookiebituryl-sugar complex;
  • the reaction temperature is not limited, but can be performed for 0 to 24 hours at 0 to 80 o C.
  • Cooker bituril may be represented by Formula 1, preferably in Formula 1, X is 0, A, and ⁇ 2 are independently H, OR, 0 (CH 2 ) aR or 0 (CH 2 ) aSR, A is an integer of 1 to 5, and R is H, C1-C30 alkyl or
  • n can be an integer from 6 to 10.
  • It may be a sugar or amino sugar antibiotic having an amino group or a salt containing the same, and specifically, may be represented by Formula 2 above.
  • the cooker bituril used in the sugar index can also add functional groups to the outward ring.
  • the present invention also provides a method for detecting sugars using cooker bituril.
  • the cookerbituril of the present invention recognizes sugars in aqueous solution and stabilizes them by supporting them.
  • the present invention is the primary molecule that melts in water without charge
  • the cucurbitur [girryl (CB [7]) provided a cucurbitur [girryl-sugar complex formed by strong linkages with aminosaccharides; the master-guest interaction was confirmed by NMR spectroscopy and MALDI-TOF mass spectrometry. NMR analysis showed that aminosaccharides exist as alpha anomers in the CB [7] cavity, and ITC analysis showed strong binding to aminosaccharides in CB [gi].
  • Ka 1.6 x 10 1
  • No artificial receptors have been reported (in the case of D-galatosamine hydrochloride) that exhibit strong binding to sugars in aqueous solutions and allow sugar stabilization of certain anomeric forms (particularly alphaanomers).
  • a cucurbitur [girryl-D-galactosamine hydrochloride complex was prepared in the same manner as in Example 1 except that D-galactosamine hydrochloride was used instead of D-glucosamine hydrochloride in Example 1.
  • D-galactosamine hydrochloride (2) is a C4-digit ⁇
  • the functional group is a C4 epimer of 1 present in the vertical direction.
  • alpha and beta anomers are present at a ratio of 63:37 after 24 hours.
  • the high field shift of the signal in the N-NMR spectrum (298 K) results in divalent CB [ It can be seen that the tile complex was formed, which can be confirmed by MALDI-TOF (FIG. 7 and FIG. 8).
  • the NMR spectrum is clearer than 1, and like 1, a better resolved signal appears when 2 equivalents of CB [71] are present.
  • Protons endocyclic methylene protons
  • Fig. 11 we can see that there is a spatial correlation between CB [Hie and H6 in tile 2 (5.4ppm), and H3 and H5 with large high field shifts of lOppm are Tell us that there is a deep common 2)-
  • hypotonic traversal is centered, i.e., the -NH 3 + (C2) and -OH (Cl) functional groups of 2 point toward the carbonyl inlet of the CB group and the OH (C6) half It means that it is located in the opposite direction.
  • the arrangement of 2 alpha anomers in CB [7] is consistent with the results of molecular mechanics modeling, indicating that Hie and H6 of 2 are in close proximity with the quantum cyclic methylene protons in the molecular model ( ⁇ 3.9 to 4.1 A).
  • oxygen atom of the ring is in the form of CB [71], stabilizing and ammonium ion through hydrogen bonding of the hydroxyl group of C 1 and C 2 of 2 and Stabilization through interaction of CB [7] carbonyl groups.
  • a cucurbitur [reel -D-galactosamine hydrochloride complex was prepared in the same manner as in Example 1 except that D-mannosamine hydrochloride was used instead of D-glucosamine hydrochloride in Example 1.
  • Example 1 neomycin or ferromycin instead of D-glucosamine hydrochloride
  • Example 1 Except for the use, the same method as in Example 1 was used to prepare cookerbitu [reel-neomycin and cookerbitu [giryl-ferromycin complex].
  • the cucurbituril of the cucurbituril-sugar complex recognizes and stabilizes the sugar, and based on this, the cucurbituril can form a complex with an aminosugar antibiotic based on this. I found out.
  • the cooker bituril of the present invention selectively binds sugars, especially amino sugars, in an aqueous solution to strongly support amino sugars in the cooker bituril joint and prevents the rotation of amino sugars. ) To stabilize.

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Abstract

본 발명은 쿠커비투릴이 당류를 인지해 쿠커비투릴 공동에 담지하고 안정화시킨 쿠커비투릴-당 복합체 및 이의 제조방법을 제공한다.

Description

명세서
발명의명칭:쿠커비투릴-당복합체및이의제조방법 기술분야
[1] 본발명은쿠커비투릴-당복합체및이의제조방법에관한것으로，보다
상세하세는쿠커비투 [n]릴이당류를인지하여공동에담지한후당류를 안정화시킨쿠커비투릴 [η]-당복합체및이의제조방법에관한것이다.
배경기술
[2] 인공당류수용체 (artificial saccharide receptor)는감지및약물전달등과같은 분야로의응용이가능해특히수용액상에서비공유결합을기반으로작용하는 인공당류수용체는생화학또는초분자화학분야에서매우흥미로운주제로 대두되고있다.
[3] 이러한당류수용체의구현에있어서，가장먼저당류와물분자의분리와그에 따른선택적인당류와의결합이라는어려움이따른다.공유결합을기반으로 하는붕산 (boronic acid)과는달리,당류인지단백질 (lectin,렉틴)과같이소수성 주머니 (hydrophobic cage)와수소결합이가능한기능기를지닌인공수용쎄는 소수에불과하다.
[4] 또한유기용매를기반으로하는수용체와는달리구조적으로，물에서
기능적으로다양한당류를인지하는수용체의구현은한계가있다.
[5] 한편아미노당류는세포의표면에대부분 N-아실레이티드 (N-acylated)형태로 존재하며,많은생물학적과정과질병의생체지표의역할을한다.또한이들은 아미노당항생물질 (aminoglycoside)의중요요소이기도하다.
[6] 그러나인공수용체의이러한잠재적인응용가능성에도불구하고아미노당류 인지에인공수용체를사용하는데에는여전히많은어려움이따른다.
[7] 앞서서술할바와같이유기용매를기반으로한수용체는매우드물며,나아가 비공유결합을기반으로하여수용액상에서작용하는인공수용체는이제껏 구현하지못하였다.
[8] 몇몇의붕산을기반으로하는수용체가존재하지만이들은아주약한결합을 보이며，금속이온역시아미노당류를인지하지만이들의웅용은감지로 제한되어있어수용액상에서비공유결합을기반으로하는인공수용체에대한 연구가절설히요구된다.
발명의상세한설명
기술적과제
[9] 본발명은글라이칸 (glycan)분석을위한인공렉틴，세포와세포의상호작용을 탐지할지질 (lipid)-CB[7]접합체및항체약물의운송과감지에유용하게 사용가능한쿠커비투릴-당복합체및이의제조방법을제공한다.
과제해결수단 [10] 본발명은초분자체인쿠커비투릴이수용액상에당류를인지하고담지하여 안정화시켜형성된쿠커비투릴-당복합체를제공하는것으로，본발명의 쿠커비투릴 -당복합체는
[11] 쿠커비투릴및，
[12] 상기쿠커비투릴의공동에담지된당을포함한다.
[13] 본발명의일실시예에따른상기쿠커비투릴은상기화학식 1로표시될수 있다.
[14] [화학식 1]
Figure imgf000004_0001
[16] [상기화학식 1에서，
[17] X는 0, S또는 NH이며，
[18] A,및八2는각각독립적으로 H, OR, SR, NHR, COOH, 0(CH2)aR또는 0(CH2)a SR이며,상기 a는 1내지 5의정수이고，상기 R은 H,할로겐， C1-C30알킬, C2-C30알케닐, C2-C30알키닐， C2-C30카르보닐알킬， C3-C30시클로알킬， C2-C30헤테로시클로알킬, C6-C30아릴， C6-C30아릴 CI-C30알킬，
C2-C30헤테로아릴또는 C2-C30헤테로아릴 C1-C30알킬이며;
[19] n은 4내지 14의정수이다.]
[20] 바람직하게본발명의일실시예에따른상기화학식 1에서 X는 0이며 , Α,및 A 2는각각독립적으로 H, OR, 0(CH2)aR또는 0(CH2)aSR이며 ,상기 a는 1내지 5의 정수이고，상기 R은 H, C1-C30알킬또는 C2-C30알케닐이며; n은 6내지 10의 정수일수있다.
[21] 본발명의일실시예에따른당은단당류,이당류또는다당류일수있으며， 바람직하게는아미노기또는이를포함하는염을가지는당일수있다.
[22] 바람직하게본발명의일실시예에따른아미노기또는이를포함하는염을 가지는당은아미노당항생제또는하기화학식 2로표시될수있다.
[23] [화학식 2]
Figure imgf000004_0002
[25] [상기화학식 2에서，
[26] Rn내지 Rl5는서로독립적으로 OH, NH2또는 NH3 +X-이며， Rn내지 Rl5중어느 하나는반드시 NH2또는 NH3 +X-이며； [27] X-는산의 1가음이온이다.]
[28] 본발명의일실시예에따른쿠커비투릴과당은당량비가 0.5내지 3 : 1일수 있다.
[29] 또한본발명은수용액상에서쿠커비투릴이당을선택적으로인지하고담지해 안정화시킨쿠커비투릴-당복합체의제조방법을제공하며，쿠커비투릴-당 복합체의제조방법은，
[30] 수용액내에서쿠커비투릴및당을접촉시켜쿠커비투릴-당복합체를제조하는 단계;를포함한다.
[31] 본발명의쿠커비투릴 -당복합체의제조방법의일실시예에따른
쿠커비투릴은상기화학식 1로표시될수있으며，바람직하게상기화학식 1에서, X는 0이며 , A,및 ^는각각독립적으로 H, OR, 0(CH2)aR또는 0(CH2 )aSR이며，상기 a는 1내지 5의정수이고，상기 R은 H, C1-C30알킬또는
C2-C30알케닐이며; n은 6내지 10의정수일수있다.
[32] 본발명의쿠커비투릴 -당복합체의제조방법의일실시예에따른당은
아미노기또는이를포함하는염을가지는당일수있으며，구체적으로 아미노당항생제또는상기화학식 2로표시될수있다.
[33] 본발명의쿠커비투릴 -당복합체의제조방법의일실시예에따른 '
쿠커비트릴과당은당량비가 0.5내지 3: 1일수있다.
[34] 또한본발명은쿠커비투릴을이용하여당을감지하는방법을제공한다. 발명의효과
[35] 본발명의쿠커비투릴 -당복합체는수용액상에서쿠커비투릴이선택적으로 당을인지하여쿠커비투릴공동에담지하고담지된당와아노머의
변광회전현상을막아안정화시킨것으로,쿠커비투릴은당류의종류에따라 특정한아노머를담지하고안정화시켜선택적인쿠커비투릴-당복합체를 형성할수있다.
[36] 또한본발명의쿠커비투릴-당복합체는쿠커비투릴외향고리의기능화를 도입하여글라이칸 (glycan)분석을위한인공렉틴또는세포와세포의
상호작용을탐지할지질 (lipid)-CB[7]접합체로의응용도가능하다.
[37] 또한본발명의쿠커비투릴-당복합체는특정항체약물의운송및감지에 사용될수있다.
[38] 또한본발명의쿠커비투릴 -당복합체의쿠커비투릴은수용액상에서당을 인지할수있어세포와세포의상호작용을탐지할수있다.
[39] 또한본발명의쿠커비투릴-당복합체의제조방법은수용액상에서
쿠커비투릴이당을인지하여쿠커비투릴공동에담지하여안정화시킨 쿠커비투릴-당복합체를용이하게제조할수있으며，본발명의쿠커비투릴-당 복합체의제조방법으로세포와세포의상호작용및항체약물의운송및감지에 적용될수있다. 도면의간단한설명
[40] 도 1은쿠커비투 [ 릴의구조를나타낸도면이며，
[41] 도 2는본발명의실시예에사용된아미노당류를나타낸도면이며，
[42] 도 3은 CB[7] . 1복합체의 NMR스펙트럼을나타낸도면이며,
[43] 도 4는 CB[7] · 1복합체의 MALDI-TOF매스스펙트럼을나타낸도면이며， [44] 도 5는 CB[7] . 1복합체와 1(아래)의 proton-coupled 13C-NMR스펙트럼을
나타낸도면이며，
[45] 도 6은 CB ]의 2당량으로형성한 CB[7] · 1복합체의 ROESY(278K)
스펙트럼을나타낸도면이며,
[46] 도 7은 CB[7] - 2복합체의 NMR스펙트럼을나타낸도면이며，
[47] 도 8은 CB[7] · 2복합체의 MALDI-TOF매스스펙트럼을나타낸도면이며， [48] 도 9는 CB[7] . 2복합체와 2(아래)의 proton-coupled 13C-NMR스펙트럼을
나타낸도면이며,
[49] 도 10은 CB[7]의 2당량으로형성한 CB[7] · 2복합체의 DQF-COSY스펙트럼을 나타낸도면이며，
[50] 도 11은 CB [기의 2당량으로형성한 CB[7] · 2복합체의 ROESY(278K)
스펙트럼을나타낸도면이며，
[51] 도 12는 B[7] · 2복합체의분자역학모델링이며,
[52] 도 13는 CB [기를첨가하였을시 2의 NMR스펙트럼을나타낸도면이며，
[53] 도 14은 298K에서물내에 CB[7](0.5mM)와 2(10mM) ITC프로파일을타나낸 것이며，
[54] 도 15은 CB [기의 2당량으로형성한 CB[7] · 3복합체의 1H NMR을나타낸 도면이며,
[55] 도 16는 298K에서물내에 CB [기와 3(10mM) ITC프로파일을타나낸것이며， [56] 도 17은 CB [기를첨가하였을시 4의 Ή-NMR스펙트럼을나타낸도면이며， [57] 도 18은 298K에서물내에 CB [기와 4(10mM) ITC프로파일을타나낸도면이다. 발명의실시를위한형태
[58] 본발명자들은당류수용체의아노머릭효과 (anomeric effect)를연구하던중 초분자체가수용액상에서당류를소수성공동 (hydrophobic cavity)에담지하여 알파아노머를안정화시킬수있을것이라예상하였으며,초분자체로 쿠커비투릴이당을인지하고담지하여안정화시키는것을발견하여
쿠커비투릴 -당복합체를제조함으로써본발명을완성시켰다.
[59] 본발명의쿠커비투릴—당복합체는
[60] 쿠커비투릴및，
[61] 상기쿠커비투릴의공동에담지된당을포함한다.
[62] 일반적으로당류는입체전자효과 (stereoelectronic effect)로인해알파
아노머 (ot-anomer)의안정화를유도하며,수용액상태에서당류는 수화작용 (hydration)으로인해베타아노머 (β-anomer)로의
변광회전현상 (mutarotation)을유도한다.
[63] 그러나본발명자들은인공수용체로초분자체인쿠커비투릴 (CB[n])이
수용액상에서선택적으로당류를인지함을발견하였으며，나아가쿠커비투릴에 담지된당의변광희전현상을막아알파아노머또는베타아노머로당을 안정화시키는것을발견하여본발명을완성하였다.
[64] 쿠커비투 [n]릴 (CB[n])은단단하고내부에소수성공동을지니는거대고리 분자이며전하를띄지않고물에녹는초분자주인분자로 CB[n]의공동과입구에 존재하는카르보닐기 (carbonyl)는소수성상호작용과강한이온-쌍극자 상호작용을기반으로안정한주인 -손님복합체의형성을가능하게하나 쿠커비투릴에당을담지되어안정화된쿠커비투릴 -당복합체는보고된적이 없었다.
[65] 본발명의쿠커비투릴 -당복합체의쿠커비투릴은수용액상에서당을인지하고 쿠커비투릴공동에담지하여안정화시킴으로써아미노글루코시드항체약물 운송및감지에적용이가능하다.
[66] 또한본발명의쿠커비투릴 -당복합체의쿠커비투릴이당을인지할수
있으므로，쿠커비투릴외향고리에기능화기를도입하여글라이칸 (glycan)분석을 위한인공렉틴또는세포와세포의상호작용을탐지할지질 (lipid)-CB[7] 접합체를제조할수있다.
[67] 본발명의일실시예에따른쿠커비투릴은하기화학식 1로표시될수있다ᅳ [68] [화학식 1]
[69] 圍 AN-CH?
Figure imgf000007_0001
X n
[70] [상기화학식 1에서,
[71] X는 O, S또는 NH이며,
[72] A,및 A2는각각독립적으로 H, OR, SR, NHR, COOH, 0(CH2)aR또는 0(CH2)a SR이며，상기 a는 1내지 5의정수이고,상기 R은 H,할로겐， C1-C30알킬， C2-C30알케닐， C2-C30알키닐， C2-C30카르보닐알킬, C3-C30시클로알킬， C2-C30헤테로시클로알킬， C6-C30아릴, C6-C30아릴 C1-C30알킬，
C2-C30헤테로아릴또는 C2-C30헤테로아릴 C1-C30알킬이며;
[73] n은 4내지 14의정수이다.]
[74] 바람직하게본발명의일실시예에따른상기화학식 1에서 X는 O이며， A,및 A 2는각각독립적으로 H, OR, 0(CH2)aR또는 0(CH2)aSR이며,상기 a는 1내지 5의 정수이고,상기 R은 H, C1-C30알킬또는 C2-C30알케닐이며; n은 6내지 10의 정수일수았다.
[75] 보다바람직하게본발명의일실시예에따른상기화학식 1에서 X는 0이며, 및 ^는각각독립적으로 H, OR이며，상기 R은 H, C1-C30알킬일수있다.
[76] 본발명에기재된본발명에기재된 「알킬」 , 「알콕시」 및그외
「알킬」 부분을포함하는치환체는직쇄또는분쇄형태를모두포함한다.또한 본발명에기재된 「아릴」 은하나의수소제거에의해서방향족
탄화수소로부터유도된유기라디칼로,각고리에적절하게는 4내지 7개， 바람직하게는 5또는 6개의고리원자를포함하는단일또는융합고리계를 포함하며，다수개의아릴이단일결합으로연결되어있는형태까지포함한다. 구체적인예로페닐,나프틸，비페닐，안트릴,인데닐 (indenyl),플루오레닐등을 포함하지만,이에한정되지않는다.본발명에기재된 ' 「헤테로아릴」 은방향족 고리골격원자로서 B, N, 0, S, P(=0), Si및 P로부터선택되는 1내지 4개의 헤테로원자를포함하고,나머지방향족고리골격원자가탄소인아릴그룹을 의미하는것으로， 5내지 6원단환헤테로아릴,및하나이상의벤젠환과축합된 다환식해테로아릴이며,부분적으로포화될수도있다.또한，본발명에서의 헤테로아릴은하나이상의해테로아릴이단일결합으로연결된형태도 포함한다.
[77] 본발명에기재된 「시클로알킬」 은포화된탄화수소고리를의미하며，
바람직하게는 5내지 7원의지환족고리일수있으며，방향족또는지환족고리가 융합된경우도포함한다.
[78] 본발명에기재된 「헤테로시클로알킬」 .은헤테로원자로서고리내에산소,황 또는질소를포함하는지환족고리를의미하며，헤테로원자의개수는 1-4이며， 바람직하게는 1-2이다.헤테로시클로알킬에서시클로알킬은바람직하게는 모노시클로알킬또는비시클로알킬이며，방향족고리린인아릴또는
헤테로아릴이융합되어있는경우도포함한다.
[79] 본발명에기재된 C1-C30알킬， C2-C30알케닐， C2-C30알키닐，
C2-C30카르보닐알킬， C3-C30시클로알킬및 C2-C30헤테로시클로알킬은 바람직하게 C1-C10알킬， C2-C10알케닐， C2-C10알키닐, C2-C10카르보닐알킬, C3-C10시클로알킬및 C2-C10헤테로시클로알킬일수있으며， C6-C30아릴및 C4-C30헤테로아릴은 C6-C12아릴및 C4-C12해테로아릴일수있다.
[80] 본발명에기재된당및당류는당업자가자명하게인식할수있는범위로
일례로단당류에서부터다당류모두를포함하는개념이며,당항생제도 포함되며，바람직하게는당항생제,단당류，이당류또는다당류일수있으며, 바람직하게는아미노기또는이를포함하는염을가지는당또는
아미노당항생제일수있으며，보다바람직하게는아미노기또는이를포함하는 염을가지는단당류및아미노기를가지는아미노당항생제일수있다.
[81] 본발명의당항생제및당항생물질은당을기반으로하는항생제및 항생물질로작용기를가지거나가지지않은항생제도포함하며， 아미노당항생제도포함한다.
[82] 본발명의아미노당항생제는아미노기를가지는당을기반으로하는
항생제들로,일례로네오마이신,페로모마이신등을들수있다.
[83] 본발명의일실시예에따른아미노기또는이를포함하는염을가지는당및 아미노당항생제는일례로， D-글루코사민염산염, D-갈락토사민염산염， 으만노사민염산염， 6-아미노ᅳ6-디옥시 -D-글루코스， N-아세틸글루코사민， 시알릭산， L-다우노사민)，네오마이신，페로모마이신을들수있으나,이에 한정되는것은아니다.
[84] 구체적으로본발명의일실시예에따른아미노기또는이를포함하는염을 가지는당은하기화학식 2로표시될수있다.
[85] [화학식 2]
Figure imgf000009_0001
[87] [상기화학식 2에서，
[88] Ru내지 Rl5는서로독립적으로 OH, NH2또는 NH3 +X-이며, Rn내지 Rl5중어느 하나는반드시 NH2또는 NH3 +X-이며；
[89] X-는산의 1가음이온이다.]
[90] 쿠커비투릴과강한결합을하기위한측면에서본발명의일실시예에따른 상기화학식 2에서 R„내지 R15는서로독립적으로 OH또는 NH3 +X-이며 , ,내지 Rl5중어느하나는반드시 NH3 +X-일수있다.
[91] 본발명의일실시예에따른상기화학식 2에서 X-는산의 1가음이온이면모두 가능하며，일례로염산이온 (C1 ),질산이은 (N03-),아세트산이온 (CH3COO)등을 들수있으며，바람직하게는염산이온일수있다.
[92] 본발명의일실시예에따른쿠커비트릴과당은몰비가 0.5내지 3 : 1일수
있으며，바람직하게는 1내지 2： 1일수있다.
[93] 또한본발명은수용액내에서쿠커비투릴및당을접촉시켜쿠커비투릴-당 복합체를제조하는단계 ;를포함하는쿠커비투릴-당복합체의제조방법을 포함한다.
[94] 구체적으로본발명의쿠커비투릴 -당복합체의제조는쿠커비투릴과당을 수용액상에녹여쿠커비투릴과당을접촉시킬수있으며，쿠커비투릴과당의 흔합하여수용액상에첨가할수있어쿠커비투릴과당을수용액상에서 접촉시키는방법이라면순서에관계없이모두가능하다.
[95] 본발명의쿠커비투릴 -당복합체의제조방법의일실시예에따른
쿠커비트릴과당은당량비가 0.5내지 3： 1일수있다.
[96] 본발명의쿠커비투릴 -당복합체의제조방법의일실시예에따른상기
b)단계의당을첨가하여쿠커비투릴-당복합체를제조하는반응시간과 반웅온도는제한이있는것은아니나， 0 ~ 80oC에서 0 ~ 24시간동안수행될수 있다.
[97] 본발명의쿠커비투릴-당복합체의제조방법의일실시예에따른
쿠커비투릴은상기화학식 1로표시될수있으며，바람직하게상기화학식 1에서, X는 0이며 , A,및八2는각각독립적으로 H, OR, 0(CH2)aR또는 0(CH2 )aSR이며,상기 a는 1내지 5의정수이고，상기 R은 H, C1-C30알킬또는
C2-C30알케닐이며; n은 6내지 10의정수일수있다.
[98] 본발명의쿠커비투릴-당복합체의제조방법의일실시예에따른당은
아미노기또는이를포함하는염을가지는당또는아미노당항생제일수있으며， 구체적으로상기화학식 2로표시될수있다.
[99] 또한당감지지표에사용되는쿠커비투릴은외향고리에기능기를도입할수 있다.
[100] 또한본발명은쿠커비투릴을이용하여당을감지하는방법을제공한다.
[101] 앞서상술한바와같이본발명의쿠커비투릴은수용액상에서당을인지하고 공동에당을담지하여안정화시킨다.
[102]
[103] 이하에서본발명의구체적인실시예를제시하는바，하기의실시예는본
발명의예시목적에불과한것으로본발명을한정하고자하는것은아니다.
[104] 본발명은전하를띄지않고물에녹는초분자주인분자인
쿠커비투 [기릴 (CB[7])이아미노당류와강한결합으로형성된쿠커비투 [기릴-당 복합체를제공하는것으로，주인 -손님상호작용은 NMR분광학과 MALDI-TOF 질량분석을통해확인하였다. NMR분석을통해아미노당류가 CB[7] 공동안에서알파아노머로존재함을확인하였으며 , ITC분석으로 CB [기이 물에서아미노당류에대해강력한결합을보임을알았다.최고의결합력은 Ka = 1.6 x 10 1 에달하며 (D-갈라토사민염산염인경우)수용액상에서당류에대해 강한결합을보이며특정아노머형태 (특히알파아노머)의당안정화를가능하게 하는인공수용체는보고된적이없다.
[105] [실시예 1]쿠커비투 [기릴 -D-글루코사민염산염 (D-glucosamine hydrochloride, 1) 복합체 (CB[7] · 1복합체)제조
[106] 실온에서물 l mL에쿠커비투릴 [7] 5.8 mg을녹여쿠커비투릴수용액을
제조하였다.여기에쿠커비투릴 [기과동일당량또는쿠커비투 [기릴대비
0.5당량의 D-글루코사민염산염을물 lmL에녹인용액을첨가하여
쿠커비투 [기릴 -D-글루코사민염산염 (D-glucosamine hydrochloride, 1)복합체를 당량비에따라각각제조하였다.
[107] D-글루코사민염산염 (D-glucosamine hydrochloride, 1)은갓 D20에녹았을때 알파아노머가과량으로존재하지만 (α:β = 93:7)시간이지남에따라
변광회전현상으로인하여 24시간후에는 65:35의비로알파와베타아노머가 존재하게된다.동일한당량의 CB[7]과 1 D20에녹인용액 (5 mM)의 Ή-NMR 스펙트럼 (Bruker DRX 500 spectrometer, 298 K)에서복합체안에서의 1의 신호 (resonance)를확인할수있으며 (도 3)，이신호의높은장이동 (upfield shift)을 통해 1이 CB[7I의공동안쪽에결합하고있음을알수있다.각신호의넓은폭은
1이 CB [ 과결합한상태와자유로운상태로의전환이빠름을보여준다.
쿠커비투 [기릴과 1의 1: 1복합체의형성은 MALDI-TOF에의해서도
확인되었다 (도 4). 2당량의 CB [기이존재함으로써， 1과 CB[7]복합체형성을 완전히이루게하고이는빠른전환을억눌러 NMR스펙트럼의신호가뚜렷하게 나타나게한다 (도 3).
[108] Ή과 l3C NMR DQF-COSY, HSQC와 ROESY스펙트럼 (278 and 298 K)을 이용하여 CB^] · 1복합체에존재하는 1을완전히분석할수있었다. 1의신호는 0.6-0.1 ppm정도의이동을보여주었으며,하나의세트로나타나고있다.예를 들면, 278K에서단하나의아노머릭신호 (anomeric peak)만존재함을불수 있으며 (4.68 ppm),이신호는자유로운상태의 1에비해높은장이동을하였다. 자유로운 1의신호들 (Η2α (3.2 ppm), Η2β (2 · 9 ppm))은복합체를이루면서하나의 신호로나타났다 (2.2 ppm).또한복합체의 proton coupled 13C-NMR스펙트럼에서 하나의이중항 (doublet)이 88.8ppm에서나타났으며이이증항의
짝지음상수 (coupling constant)는 = 170.4 Hz이다 (도 5).이값들은자유로운 상태의 1의알파아노머의화학적이동과짝지음상수 (88.5 ppm,쒜
=170.5Hz)와흡사한값이며이는 1이복합체안에서알파아노머로존재함을 암시한다.더불어，복합체의 ROESY스펙트럼 (도 6)에서는 Hle-H2«(l)의 상관관계점적 (correlation spot)이나타나는반면 Η3α혹은 Η5α이 Ηΐα과이루는 어떠한상관관계점적이보이지않아 1의 C1-OH가수직방향으로존재하여알파 아노머의구조를가짐을확인할수있다.흥미롭게도， ROESY스펙트럼에서 CB [기과 1분자사이의약한교차신호 (cross-peak)를볼수있다.종합적인 NMR 분석은 CB [기이 1의알파아노머를안정화시킴을시사한다.
[109] 신호의폭넓음으로인하여 Ή-NMR적정을통한 1과 CB [기의결합상수의
정량적인측정이어렵기때문에등온열량측정기 (isothermal titration calorimetry, ITC)를이용하여복합체형성의결합상수를측정하였다. ITC(VP-ITC, Microcal, Inc)실험결과 (표 1) CB [기이 1과강한결합을보이는것을확인하였으며 (Ka = (4.4 ± 1.0)χ 103Μ-')이들의결합이엔탈피와엔트로비의변화가선호하는 방향으로이루어짐을알수있다 (ΔΗ0 = -14.0 KJ mol-^TAS0 = 6.6KJmo ).이러한 변화에대한설명은아래에서논의하도록하겠다.
[110] 표 1 [Table 1 ]
Figure imgf000012_0001
[111] 표준오차는세번의독립적인 ITC적정실험을통해얻어졌다ᅳ
[112]
[113] [실시예 ¾쿠커비투 [7]릴 -D-갈락토사민염산염 (D-galactosamine hydrochloride, 2 )복합체 (CB[7] · 2복합체)제조
[114] 실시예 1에서 D-글루코사민염산염대신 D-갈락토사민염산염를사용한것을 제외하고는실시예 1과동일한방법으로쿠커비투 [기릴 -D-갈락토사민염산염 복합체를제조하였다.
[115] 으갈락토사민염산염 (D-galactosamine hydrochloride, 2)은 C4자리의 ΟΗ
기능기가수직방향으로존재하는 1의 C4에피머 (epimer)이다. 2역시 D20에서 변광회전현상으로인하여 24시간후에는 63:37의비로알파와베타아노머가 존재하게된다.또한 Ή-NMR스펙트럼 (298 K)에서신호의높은장이동을통해 2 가 CB [기와복합체를형성하였음을알수있으며이는 MALDI-TOF를통해서도 확인할수있다 (도 7도 8). 2의경우 1에비해 NMR스펙트럼이또렷하고, 1과 마찬가지로 2당량의 CB[71이존재할때더욱더잘분해된신호를나타낸다. 하나의아노머신호의존재는 CB [기이하나의아노머만을담지하고있음을 보이며, proton-coupled l3C-NMR에서나타나는하나의이중항 (88.8 ppm, '7c l.Hi = 170.7Hz)은 CB ]의공동에자리잡은 2가알파아노머로존재하고있음을 시사하고있다 (도 9).게다가， DQF-COSY에서수평방향으로향해있는
Hl(Hle)가나타내는짝지음상수 (ν^Η^^πίζΛ/Η^Η^ΙΟ/ΖΗζ)를얻을수있으며 이를통해알파아노머의존재를확인할수있다 (도 10).마찬가지로 ROESY 역시 Hl e-H2a의상관관계이외의다른교차신호를보이지않는다 (도 U).
종합적인 NMR분석을통해 CB[7]공동안에서 2가알파아노머로존재함을 확인하였다.
[116] 일반적으로손님분자의첨가로인한 CB [기의메틸렌양성자 (methylene
proton)의장이동은일어나지않지만，카르보닐입구의고리안메틸렌
양성자 (endocyclic methylene protons)는손님분자와가까이존재하며그들사이의 공간적상관관계 (through space correlation (< - 5人))를 ROESY스펙트럼을통해 확인할수있다. ROESY스펙트럼 (도 11)에서 CB [기과 2의 Hie그리고 H6사이에 공간적상관관계가존재함을확인가능하며 (5.4ppm), l .Oppm의큰높은장 이동을한 H3과 H5은그들이 CB[7]공동깊숙이존재한다는것을말해준다 (표 2)-
[1 17] 표 2
[Table 2]
Figure imgf000013_0001
[1 18] 이는 2가 CB [ 의공동안에서핵사머릭링 (hexameric ring)의 C2와 C6를
가로지르는가상의긴축을중심으로삐뚤게자리잡았음을시사한다.즉, 2의 -NH3 +(C2)와 -OH(C l)기능기가 CB [기의카르보닐기입구를향하고있으며 OH(C6)은반대방향으로위치함을뜻한다. CB[7]공동안에서의 2의알파 아노머의배치는분자역학모델링 (molecular mechanics modeling)결과와 일치하는데，분자모델에서 2의 Hie와 H6가고리안메틸렌양성자와근접해 있음을 (~ 3.9 to 4.1 A)보여준다 (도 12).또한고리의산소원자가 CB[71의공동에 물힌형태로， 2의 C 1과 C2의하이드록시기 (hydroxyl group)의수소결합을통한 안정화와암모늄이온 (ammonium ion)과 CB[7]카르보닐기의상호작용을통한 안정화를제시한다.
[119] 신호의폭넓음과변광회전현상으로인하여 Ή ΝΜΙ 적정을통한 CBm과 2 사이의결합력측정이어렵다 (도 1 3).이에 ITC분석을통하여 (표 1 ,도 14) CB [기이 2에택해강한결합력을보임을확인할수있었으며 ( a=(1 .6 ± 0.1 )x l04 M-i)이결합력은이전의어떠한물속에서작용하는당류인공수용체도보이지 못한값이다.이들의결합역시 1과마찬가지로엔탈피와엔트로피변화가 선호하는방향으로 (Aff" = - 13.8 kJ mol-',TAS" = 10.1 kJ mol 1)진행되었다.엔탈피 변화가거의비슷한반면 1에비해서 2는높은엔트로피변화를보이는데， 복합체를형성과정에서일어나는수화걸층 (hydration shell)의방출이 2에서더 강하게일어나며，이것이강한결합이가능하게하는요인이라사료된다.
[120] CBm의카르보닐입구와아미노당류의암모늄이은이이루는이온-쌍극자 상호작용을기반으로하는주인손님상호작용은상대적으로큰음의엔탈피 변화를나타낸다.또한양의엔트로피변화로보여지는소수성상호작용역시 중요한역할을하는것으로보인다ᅳ CB [기의공동에는높은에너지를가지는물 분자가존재하는데,이들은손님분자 (1,2)의결합으로통해방출된다.이들은 엔트로피의변화뿐만아니라엔탈피의변화에도기여한다.이와같이물을 매개로한당류와렉틴의결합은생물학적인조건에서도많이발견되지만， 단당류의알파아노머를수용성초분자의소수성공동안에안정화시키는것은 보고된적이없다.
[121]
[122] [실시예 3]쿠커비투 [기릴 -D-만노사민염산염 (D-mannosamine hydrochloride, 3) 복합체 (CB[7] . 3복합체)제조
[123] 실시예 1에서 D-글루코사민염산염대신 D-만노사민염산염를사용한것을 제외하고는실시예 1과동일한방법으로쿠커비투 [ 릴 -D-갈락토사민염산염 복합체를제조하였다.
[124] 순차적으로， C2위치에수직배향하는암모늄이온을지닌 D-만노사민
염산염 (D-mannosamine hydrochloride, 3)과 CB [ 의결합에대한연구를
진행하였다. 3역시 D20에서변광회전현상을보이며알파와베타의비가
29:기에서 24시간이후 38:62로변하였다. CB [기과 3의 2: 1흔합물의 1
H-NMR스펙트럼 (도 15)에서복합체의형성으로 3의신호가높은장이동을함을 확인하였다.그러나， 1과 2와는달리두개의아노머의존재로인해스펙트럼이 복잡하다.면밀한 NMR분석으로아노머릭비율 (anomeric ratio)가복합체의 형성으로알파아노머를선호하는방향 (α:β = 65:35)으로바뀌었음을볼수있다. ITC실험으로복합체의형성 (/ς=(1.9 ± 0.3)χ Κ Μ-1)이엔탈피와엔트로피의- 변화를통해이루어짐을확인하였으며 (AHU = -13.0 kJ mol-1 ΓΔ5" = 5.7 kJ mol 1), 1 : 1주인 -손님결합모델을이용하여얻은 N값은 -0.75이다 (표 1,도 16).이는 C2 자리에존재하는수직의 -ΝΗ3 +로인하여 CB [기공동안에서 3의배향에영향을 주어고리산소가주변의물에노출되고변광회전현상이가능해진것으로 보인다.
[125]
[126] [실시예 4]
[127] 쿠커비투「71림- D-6-아미노 -6-디옥시 -D-금루코스 (ᅳ 6-amjno-6-deoxy-D-glucose. 4) 복합체 (CB I * 4복합체ᅵ제조
[128] 실시예 1에서 D-글루코사민염산염대신 D-6-아미노 -6-디옥시 -D-글루코를
사용한것을제외하고는실시예 1과동일한방법으로
쿠커비투 [기릴 -D-갈락토사민염산염복합체를제조하였다.
[129] 동일한당량의 CB ]과 4의흔합물의 Ή-NMR스펙트럼 (도 17)에서손님분자 4 의신호가높은장이동을한것을볼수있으며， 2당량의 CB ]이존재할때 더욱선명한신호를볼수있다.면밀한 NMR분석으로통해 4역시 CB[7] 공동안에알파아노머로서존재함을확인할수있었다.또한 ITC실험으로 CB [기이 4와강한결합을보임을알았다 (/ς=(1.6 ± 0.7)χ 103Μ-',표 1，도 18).
[130]
[131] [실시예 5]쿠커비투 [기릴 -네오마이신및쿠커비투 [기릴-페로모마이신복합체 제조
[132] 실시예 1에서 D-글루코사민염산염대신네오마이신또는페로모마이신를
사용한것을제외하고는실시예 1과동일한방법으로쿠커비투 [ 릴 -네오마이신 및쿠커비투 [기릴-페로모마이신복합체를제조하였다.
[133]
[134] 하기표 3에서보이는바와같이 ITC적정으로 CB [기이네오마이신 (neomycin)과 패로모마이신 (paramomycin)에강하게결합하는것을알수있다. 표 3
[Table 3]
Figure imgf000015_0001
[136] 본발명의실시예 1내지 4에서보이는바와같이쿠커비투릴-당복합체의 쿠커비투릴이당을인지하고안정화시키는것을알수있었으며，이를기반으로 쿠커비투릴이아미노당항생물질과복합체를형성할수있는지를실시예 5에 알아보았다.
[137] 본발명의쿠커비투릴 -당복합체의쿠커비투릴은수용액상에서선택적으로 당,특히아미노당류와강하게결합하여쿠커비투릴공동에아미노당류를 담지하고아미노당류의변광회전현상을막아특정아노머 (특히알파아노머)를 안정화시킨다.
[138] 또한실시예 5에서보이는바와같이쿠커비투릴이네오마이신또는
꿰로모마이신과같은아미노당항생물질을인지하고담지함을알수있었으며， 이로써본발명의쿠커비투릴-당복합체는항체약물의운송및감지에도 유용하게사용될수있음을알수있다.

Claims

청구범위
[청구항 1] 쿠커비투릴및,
상기쿠커비투릴의공동에담지된당을포함하는쿠커비투릴-당 복합체.
[청구항 2] 제 1항에있어서，
상기쿠커비투릴은상기화학식 1로표시되는것인쿠커비투릴-당 복합체.
Figure imgf000016_0001
[상기화학식 1에서，
X는 0, S또는 NH이며,
A,및 A2는각각독립적으로 H, OR, SR, NHR, COOH, 0(CH2)aR 또는 0(CH2)aSR이며,상기 a는 1내지 5의정수이고,상기 R은 H, 할로겐， C 1 -C30알킬， C2-C30알케닐, C2-C30알키닐,
C2-C30카르보닐알킬, C3-C30시클로알킬,
C2-C30해테로시클로알킬, C6-C30아릴， C6-C30아릴 C1-C30알킬,
C2-C30헤테로아릴또는 C2-C30헤테로아릴 C1-C30알킬이며; n은 4내지 14의정수이다.]
[청구항 3] 제 2항에있어서,
상기화학식 1에서, X는 0이며,
A,및 A2는각각독립적으로 H, OR, 0(CH2)aR또는 0(CH2
)aSR이며,상기 a는 1내지 5의정수이고，상기 R은 H, C1-C30알킬 또는 C2-C30알케닐이며;
n은 6내지 10의정수인쿠커비투릴 -당복합체.
[청구항 4] 제 1항에있어서，
상기당은아미노기또는이를포함하는염을가지는당인 쿠커비투릴 -당복합체.
[청구항 5] 제 4항에있어서,
상기당은단당류,이당류또는다당류인쿠커비투릴-당복합체.
[청구항 6] 제 4항에있어서，
상기아미노기또는이를포함하는염을가지는당은
아미노당항생제또는하기화학식 2로표시되는것인 쿠커비투릴 -당복합체.
Figure imgf000017_0001
[상기화학식 2에서,
Rn내지 Rl5는서로독립적으로 OH, NH2또는 NH3 +X-이며， Ru 내지 Rl5중어느하나는반드시 NH2또는 NH3 +X-이며;
X-는산의 1가음이온이다.]
[청구항 7] 제 I항에있어서,
상기쿠커비트릴과당은당량비가 0.5내지 3: 1인쿠커비투릴-당 복합체.
[청구항 8] 수용액내에서쿠커비투릴및당을접촉시켜쿠커비투릴-당
복합체를제조하는단계 ;를포함하는쿠커비투릴-당복합체의 제조방법.
[청구항 9] 제 8항에있어서，
상기쿠커비투릴은상기화학식 1로표시되는쿠커비투릴-당 복합체의제조방법.
Figure imgf000017_0002
[상기화학식 1에서,
X는 0, S또는 NH이며,
A,및八2는각각독립적으로 H, OR, SR, NHR, COOH, 0(CH2)a 또는 0(CH2)aSR이며,상기 a는 1내지 5의정수이고，상기 R은 H. 할로겐， C1-C30알킬， C2-C30알케닐, C2-C30알키닐,
C2-C30카르보닐알킬, C3-C30시클로알킬,
C2-C30헤테로시클로알킬， C6-C30아릴， C6-C30아릴 C1-C30알킬
C2-C30헤테로아릴또는 C2-C30해테로아릴 C1-C30알킬이며; n은 4내지 14의정수이다.]
[청구항 10] 제 9항에있어서,
상기화학식 1에서, X는 o이며，
Aᅳ및 A2는각각독립적으로 H, OR, 0(CH2)aR또는 0(CH2 )aSR이며,상기 a는 1내지 5의정수이고，상기 R은 H, C1-C30알킬 또는 C2-C30알케닐이며;
n은 6내지 10의정수인쿠커비투릴 -당복합체의제조방법.
[청구항 11] 제 8항에있어서，
상기당은아미노기또는이를포함하는염을가지는당인 쿠커비투릴 -당복합체의제조방법.
[청구항 12] 제 1 1항에있어서，
상기아미노기또는이를포함하는염을가지는당은
아미노항생제또는하기화학식 2로표시되는것인쿠커비투릴-당 복합체의제조방법.
Figure imgf000018_0001
[상기화학식 2에서，
Ru내지 Rl5는서로독립적으로 OH, NH2또는 NH3 +X-이며， Ru 내지 R15중어느하나는반드시 NH2또는 NH3 +X-이며；
X-는산의 1.가음이온이다.]
[청구항 13] 제 8항에있어서，
상기쿠커비트릴과당은당량비가 0.5내지 3 : 1인쿠커비투릴-당 복합체의제조방법. '
[청구항 14] 쿠커비투릴을이용하여당을감지하는방법.
PCT/KR2014/011834 2013-12-04 2014-12-04 쿠커비투릴-당 복합체 및 이의 제조방법 Ceased WO2015084075A1 (ko)

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