A kind of organic base catalytic sulfydryl-alkynyl reaction builds multi-functional small molecules probe
Method
Technical field
Present invention relates particularly to the method that a kind of organic base catalytic sulfydryl-alkynyl reaction builds multi-functional small molecules probe,
Belong to molecular image and molecular probe field.
Background technology
Molecular imaging (molecular imaging), as an emerging subject, is to use iconography means, passes through
Non-invasive mode can obtain expression and activity (such as protease and protein kinase) and apoptosis, the blood of specific molecular
The information of the physiological process such as pipe generation and transfer, and these information can be the early diagnosis of cancer, personalized treatment and medicine
Thing research and development provide corresponding and help.Molecular imaging is proposed by Harvard University Ralph professor Weissleder the earliest, this subject
Combine multiple subject technologies such as nuclear medicine, radiological medicine, chemistry, chemical biology, molecular biology and nano science.Molecule
Image technology has become as indispensable instrument in diagnosis now.
Conventional molecular image technology includes NMR (Nuclear Magnetic Resonance)-imaging (Magnetic resonance imaging, MRI), meter
Calculation machine x-ray tomography imaging (CT), positron emission computerized tomography imaging (positron emission tomography,
PET), single photon emission computed tomography (single photon emission computed tomography,
SPECT), optical imagery (including multi-photon imaging, near-infrared one district's imaging, near-infrared two district's imaging and co-focusing imaging etc.) and
Ultra sonic imaging.Every kind of imaging technique is respectively arranged with pluses and minuses at aspects such as sensitivity, principle, resolution and acquisition times.
The various imaging pattern of table 1 compares
But there is no single image technology at present to possess all of advantage and provide detection object most complete
Information, therefore research worker begins attempt to the combination of different modalities image technology, because the combination of different modalities imaging technique is not
It is only capable of the shortcoming overcoming single mode imaging technique, and the advantage of different molecular image technology can be complementary to one another, produce
Cooperative effect can be detection object provide more comprehensively, information more accurately, the combination of such as PET-NIRF image technology,
The high sensitivity of PET can make up the deficiency of optics, and the spatial resolution that optics then can make up PET is the highest.Therefore when this
After planting the combination of bimodal imaging technique, having the advantages such as high spatial resolution, rapid feedback, deep tissue infiltration, this is without being suspected to have
Help improve the accuracy rate of tumor imaging diagnosis.
The core of molecular image is to obtain high-quality, the image of special target molecule in real time, and therefore it needs to design various
The molecular probe that targeting is good, tumor uptake is high, imaging signal to noise ratio is high, to meet the demand of clinical research.And multi-modal molecule
Probe than single mode molecular probe can for detection object provide more comprehensively with accurate information.Therefore polyfunctional molecule probe
Design and preparation become most active research field in molecular image subject in recent years.The core building polyfunctional molecule probe is asked
Topic is how to be incorporated into same by an effective chemical tools by several functions chemoattractant molecule (reporter molecules and targeted molecular)
On molecular probe skeleton.The molecule platform reported at present includes: little molecule platform;High molecular polymer platform, and nanometer
Material platform.Each platform is owned by respective pluses and minuses.Currently, build the most multi-functional by easy chemical method
Molecular probe is still a challenging job.
Small-molecule probe is still the optimum selection of clinical practice at present.Small-molecule probe is generally by liver and renal system
Metabolism, checkout time is shorter, thus avoids xicity related due to be retained in that liver and kidney produce for a long time.Although it is multi-functional little
Molecular probe is simplest in theory, but preparation is got up the most difficult.This is also to cause putting down relative to other molecules
Platform, one of relatively small number of reason of report of multi-functional small molecules probe.Up to the present, multi-functional little point of the structure reported
Son method include: based on Cyclen-1,4,7,10-tetraacethyl (Bio.Med.Chem.Lett,
2011,21,3423-3426), Isosorbide-5-Nitrae, 8,11-tetraazacyclododecanes [6,6,2] hexadecane (Angew.Chem.Int.Ed, 2009,48,
7346-7349) and cyanuric trichloride molecule platform (Bioconjugate.Chem, 2014,25,761-772) equimolecular platform structure
Build.But the construction method of these current multi-functional small molecules probes deposits following a few point defect: 1) synthesis step is the most loaded down with trivial details
(multistep synthesizes and is usually directed to the step of upper protection-deprotection group);2) severe reaction conditions, poor selectivity, and lead to
Often can produce several isomerized products.The most how to develop effective molecular tool and build multi-functional small molecules probe efficiently still
It is a challenging job.
Click chemistry is a kind of novel method for synthesizing grown up in recent years, the advantage numerous due to it and by extensively
Pay close attention to, and the synthesis of guide's compound library, proteomics, Bioconjugation technology, the crowd such as materials chemistry and biological medicine
Multi-field obtain development at full speed.In numerous click chemistries, sulfydryl-alkynyl click chemistry beyond doubt of greatest concern, two
Molecule sulfhydrylation functional molecular under the conditions of photocatalysis etc. in the importing alkynyl structure of controllable high-efficiency, the gentle (room of this reaction condition
Temperature and water are as reaction dissolvent), productivity is high and bio-compatibility is high, and tolerates various active group (NH2, COOH,
OH), therefore sulfydryl-alkynyl click chemistry is successfully applied to build multifunctional material such as multifunctional macromolecule etc..But,
Using light-catalysed method to be very difficult to build some photaesthesia molecules (such as some fluorescent small molecule probes), photocatalysis simultaneously is very
The easily a series of by-product of induction sulfydryl generation: such as disulfide, therefore explores the sulfydryl-alkynes under novel catalyst
Base reacts, and is highly important.The most so far there is not yet multi-functional little point built based on sulfydryl-alkynyl click chemistry
The document report of sub-probe and patent application.
Summary of the invention
The technical problem to be solved is to provide a kind of organic base catalytic sulfydryl-alkynyl reaction to build multi-functional
The method of Small-molecule probe.
The inventive method can overcome existing photocatalysis sulfydryl-alkynyl click chemistry reaction system to be applied to build many merits
The deficiency of energy light-sensitive material, develops a kind of novel mercapto-alkynyl catalyst system and catalyzing, can be used for building the multi-functional identification of specificity and examines
Multi-modal and many targeting Small-molecule probe in disconnected reagent.
The technology path that the present invention provides is as follows:
R in above-mentioned route1It is the group after hereinafter compound R 1 removes hydrogen, the R in above-mentioned route2、R3It is hereafter respectively
Middle compound R 2, R3 remove the group after-SH.
Specifically comprise the following steps that
1) take compound Y and enter in reaction vessel, under nitrogen protection, add dimethyl sulfoxide solvent, then add chemical combination
Thing R1 and BTA-N, N, N, N-tetramethylurea hexafluorophosphate, room temperature reaction 6-12h, reaction is purified after terminating,
I.e. obtain intermediate 1;
2) take compound 1 and add in reaction vessel, under nitrogen protection, add water and dimethyl sulfoxide mixed solvent, then
Adding compound R 2 and organic base, room temperature reaction 6-24h, reaction is purified after terminating, i.e. obtains intermediate 2 and 3;
3) take compound 2 and add in reaction vessel, under nitrogen protection, add water and dimethyl sulfoxide mixed solvent, then
Adding compound R 3 and organic base, room temperature reaction 6-24h, reaction is purified after terminating, i.e. obtains compound 4.
When step 2) described in compound 1, compound R 2, the mol ratio of organic base be when being 1:1.2:0.15, intermediate
Product is 2;When described compound 1, compound R 2, the mol ratio of organic base are 1:2.5:0.15, midbody product is 3.
Step 3) described in compound 2, compound R 3, the mol ratio of organic base be 1:1.2:0.15.
Described water and the mixed solvent of dimethyl sulfoxide, the concentration of dimethyl sulfoxide is 0.2M.
The present invention also provides for another kind of method, specific as follows:
A) take compounds X and enter in reaction vessel, under nitrogen protection, add dimethyl sulfoxide solvent, then add chemical combination
Thing R1 and N, N-diethyl isopropyl amine, room temperature reaction 6-12h, reaction is purified after terminating, i.e. obtains intermediate 5;
B) take compound 5 and add in reaction vessel, under nitrogen protection, add water and dimethyl sulfoxide mixed solvent, then
Adding compound R 2 and organic base, room temperature reaction 6-24h, reaction is purified after terminating, i.e. obtains intermediate 6 and 7;
C) take compound 6 and add in reaction vessel, under nitrogen protection, add water and dimethyl sulfoxide mixed solvent, then
Adding compound R 3 and organic base, room temperature reaction 6-24h, reaction is purified after terminating, i.e. obtains compound 8.
When the compound 5 described in step b), compound R 2, the mol ratio of organic base are 1:1.2:0.15, intermediate
Product is 6;When described compound 5, compound R 2, the mol ratio of organic base are 1:2.5:0.15, midbody product is 7.
Compound 6 described in step c), compound R 3, the mol ratio of organic base are 1:1.2:0.15.
Described water and the mixed solvent of dimethyl sulfoxide, the concentration of dimethyl sulfoxide is 0.2M.
Wherein said organic base is N, N-Diisopropylethylamine (DIPEA),
1,8-Diazabicyclo[5.4.0]undec-7-ene(DBU),Triethylamine(Et3N),
1,5,7-Triazabicyclo [4.4.0] dec-5-ene (TBD), quinine, triethanolamine, ammonia or pyridine etc..
Wherein R1 is target polypeptide and antibody, including: RGD (includes monomer and dimer), and AE105 (includes 8-
The AE105 that aminocaprylic acid modifies, and the AE105 of the PEG modification of various length), RM26 (includes 8-
The RM26 that aminocaprylic acid modifies, and the RM26 of the PEG modification of various length), Knottins, affibody (parent
And body), antibody (antibody).
Wherein R2, R3 can stand alone as any one structure following:
N is the integer of 0-18.
Compared with prior art, the invention have the advantage that 1. present invention use the catalytic condition of organic base to replace traditional
Photocatalysis, is possible not only to for building light activated multifunctional optical probe, can avoid the generation of disulfide simultaneously.2.
Preparation method of the present invention is simple, can be by the equivalent of thiol molecule, and controlled realizes monosubstituted and disubstituted product.3. the present invention
Can complete in aqueous phase system, organic base is with low cost simultaneously, environmentally safe.
Accompanying drawing explanation
Fig. 1 is that organic base catalytic builds near-infrared two district multi-modal Small-molecule probe SCH1 mass spectral results.
Fig. 2 is that organic base catalytic builds near-infrared two district multi-modal Small-molecule probe SCH1 transmitting wavelength.
Fig. 3 is that the NIR-II/PET of organic base catalytic structure near-infrared two district multi-modal Small-molecule probe SCH1 is multi-modal aobvious
Picture.
Fig. 4 is absorbing wavelength and the transmitted wave that organic base catalytic builds near-infrared one district multi-modal Small-molecule probe CHS1
Long.
Fig. 5 is that the NIR-I/PET of organic base catalytic structure near-infrared one district multi-modal Small-molecule probe CHS1 is multi-modal aobvious
Picture.
Detailed description of the invention
Embodiment 1: synthesis SCH1
Raw material Y (0.7mg, 0.01mmol) and RGD-PEG is weighed in 2mL test tube3(13.9mg, 0.008mmol) is dissolved in
In DMF solvent, add the HBTU of 2equiv.After room temperature reaction 12 hours.Reactant liquor is directly cold with preparing HPLC vacuum after purification
Freezing the product being dried to obtain is white solid 5a, 10.00mg, yield 58%.MS value of calculation: C81H117N23O24 +([M+H]+):
1796.8, measured value: ESI-MS:m/z1796.5.
In 2mL test tube, weigh 5a (1.79mg, 1 μm ol) and SY1 (0.79mg, 1.5 μm ol) be dissolved into DMSO/H2O(c
=0.2M) in mixed solvent, add DIPEA (0.2equiv).After reacting 1 hour under room temperature.Reactant liquor is directly with preparing HPLC
It is product 6a that purification lyophilization obtains white powder, 0.863mg, yield 43%.MS value of calculation: C103H151N28O30S2 +([M+
H]+): 2324.0, measured value: ESI-MS:m/z2324.6.
6a (2.32mg, 1 μm ol) and CH1055SH (1.54mg, 1.5 μm ol, 1.5equiv) is weighed molten in 2mL test tube
Solve DMSO/H2In O (c=0.2M) mixed solvent, add DBU (0.2equiv).After reacting 6 hours under room temperature.Reactant liquor is straight
Connect and purify lyophilization to obtain blue powder be product SCH1 with preparing HPLC, 1.02mg, yield 30%.MALDI-TOF calculates
Value: C159H1979N35O37S5: 3350.8, measured value: ESI-MS:m/z 3350.2.
Embodiment 2: synthesis 2a
2mL test tube weighs raw material X (1.5mg, 0.005mmol) and AE105 (6.0mg, 0.004mmol,
0.8equiv) it is dissolved in DMF solvent, adds the DIPEA of 2equiv.After room temperature reaction 5 hours.Reactant liquor is directly with preparing HPLC
The product that vacuum lyophilization obtains after purification is white solid 1a, 4.02mg, yield 58%.MS value of calculation: C81H114N17O19 +
([M+H]+): 1628.8, measured value: MALDI-MS:m/z 1629.3.2mL test tube weighs 1a (1.63mg, 1 μm ol) and
SY1 (0.79mg, 1.5 μm ol, 1.5equiv) is dissolved into DMSO/H2In O (c=0.2M) mixed solvent, add DIPEA
(0.15equiv).After reacting 6 hours under room temperature.Reactant liquor is directly purified lyophilization and is obtained white powder and be with preparing HPLC
Product 2a, 0.863mg, yield 43%.MALDI-MS value of calculation: C103H147N22O25S2 +([M+H]+): 2156.0, measured value:
2156.7。
Embodiment 3: synthesis 3a
X (2.9mg, 0.01mmol) and c (RGDfK) (3.01mg, 0.005mmol, 1.0equiv) is weighed in 2mL test tube
It is dissolved in DMF (DMF) solvent, adds the DIPEA (DIPEA) of 2 equivalents.Room temperature reaction 6
After hour, reactant liquor is directly white solid 1b, 2.22mg with preparing the product that HPLC vacuum lyophilization after purification obtains, and receives
Rate 57%.ESI-MS value of calculation: C38H54N9O9 +([M+H]+): 780.4, measured value: 780.7.
2mL test tube weighs 1b (0.8mg, 1 μm ol) and SY2 (2.76mg, 4 μm ol, 4.0equiv) and is dissolved into DMSO/
H2In O (c=0.2M) mixed solvent, add DBU (0.15equiv).After reacting 18 hours under room temperature.Reactant liquor is directly with preparing
HPLC lyophilization after purification obtains white powder 3a, 0.55mg, yield 23%.MALDI-MS value of calculation: C98H144N27O26S2 +
([M+H]+): 2179.0, measured value: 2179.4
Embodiment 4: synthesis 4a
2a (2.158mg, 1 μm ol) and mercaptoethylmaine (0.156mg, 2 μm ol, 2.0equiv) are dissolved into by 2mL test tube
DMSO/H2In O (c=0.2M) mixed solvent, add DBU (0.15equiv).After reacting 8 hours under room temperature.Reactant liquor is directly used
Preparation HPLC lyophilization after purification obtains white powder SY3,0.80mg, yield 36%.MALDI-MS value of calculation:
C105H154N23O25S3 +([M+H]+): 2233.1 measured values: 2233.5
In 2mL test tube by SY3 (1.111mg, 0.5 μm ol) and Cy5.5 succinimide ester (0.785mg, 1 μm ol,
2.0equiv) it is dissolved into DMSO/H2In O (c=0.2M) mixed solvent, add DBU (0.15equiv).React 4 hours under room temperature
After.Reactant liquor directly obtains blue powder 4a, 0.532mg, yield 38% with preparing HPLC lyophilization after purification.MALDI-MS
Value of calculation: C145H194N25O26S3 +([M+H]+): 2797.4 measured values: 2798.1.