* All experimental methods are cited from the reference, please refer to the original source for details. We do not guarantee the accuracy of the content in the reference.
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 20℃; for 2 h;
To the solution of 3,3'-dithioldipropionic acid (1 g, 4.75 mmol) in dichloromethane (20 mL), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC) (2.16 g, 10.5 mmol) and N-hydroxysuccinimide (1.21 g, 10.5 mmol) were added. The mixture was stirred for 2 h at room temperature. The solution was concentrated in vacuo, and the obtained residue was subjected to silica column chromatography (ethyl acetate/hexane = 1:5) to give the NHS ester 2 (1.63 g, 85percent). 1H NMR (DMSO-d6, 400 MHz) δ 3.16 (m, 8H), 2.82 (s, 8H). FAB (DMSO-d6) [(M+H)+] calcd 405, found 405.
70%
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 20℃;
One kind of bis(succinimidyl) 3,3'-dithiodipropionate synthesis, the following steps: -; Weigh 3,3'-thiodipropionic acid (6.0g, 0.028mol), N-hydroxysuccinimide (8.0g, 0.06mol), EDC hydrochloride (13g, 0.06 mol), into a 100mL round bottom flask, 40mL of methylene chloride was added, stirred at room temperature, the solid gradually dissolved overnight reaction, there is generated a white solid. Filtered and dried to give the product (8.05g, white solid), yield 70percent.
49%
With dicyclohexyl-carbodiimide In dichloromethane; acetone at 20℃; for 24 h;
. 3,3'-Dithiobis(propanoic acid) (9.5 g, 45.2 mmol) was dissolved in a mixture of acetone (600 mL) and dichloromethane (600 mL) with stirring at room temperature. N-Hydroxysuccinimide (12.68 g, 110.2 mmol, 2.44 eq) was dissolved in the solution, then 1,3-dicyclohexylcarbodiimide (25.9 g, 125.5 mmol, 2.78 eq) was cautiously added. The mixture was allowed to stir at room temperature for 24 hours, after which time the solution was vacuum filtered and the residual solid discarded. The solvent was removed from the filtrate under vacuum, and the oily residue was redissolved in dichloromethane (ca 200 mL). The solution was reduced in volume (ca. 50 mL) and cooled, giving the product as a colourless, crystalline solid (8.90 g, 49percent yield). 1H-NMR (d6-DMSO): δ 2.80 (s, 8H), 3.05 (m, 8H). 13C-NMR (d6-DMSO): δ 25.8, 30.7, 32.3, 167.9, 170.4 ppm.
Reference：
[1] Bioorganic and Medicinal Chemistry, 2012, vol. 20, # 1, p. 96 - 100
[2] Organic and Biomolecular Chemistry, 2005, vol. 3, # 12, p. 2255 - 2261
[3] Patent: CN105315191, 2016, A, . Location in patent: Paragraph 0021; 0022; 0023
[4] Patent: US7074766, 2006, B1, . Location in patent: Page/Page column 44
[5] Angewandte Chemie - International Edition, 2010, vol. 49, # 26, p. 4405 - 4408
[6] Organic Letters, 2007, vol. 9, # 6, p. 1009 - 1012
[7] Rapid Communications in Mass Spectrometry, 2013, vol. 27, # 1, p. 238 - 248
[8] Patent: CN104667297, 2017, B, . Location in patent: Paragraph 0056-0058
[9] Nanomedicine: Nanotechnology, Biology, and Medicine, 2018, vol. 14, # 4, p. 1123 - 1136
2
[ 74124-79-1 ]
[ 1119-62-6 ]
[ 57757-57-0 ]
Reference：
[1] Journal of Mass Spectrometry, 2011, vol. 46, # 1, p. 1 - 11
3
[ 6066-82-6 ]
[ 107-96-0 ]
[ 57757-57-0 ]
Reference：
[1] Chemistry - A European Journal, 2014, vol. 21, # 10, p. 3956 - 3967
3-[2-(2-{2-[4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-phenoxy]-acetylamino}-ethylcarbamoyl)-ethyldisulfanyl]-propionic acid 2,5-dioxo-pyrrolidin-1-yl ester[ No CAS ]
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃; for 2h;
General procedure: Organic synthesisGeneral procedure for the synthesis of linker molecules (compound 1 and 2)To a solution of the di-acid (suberic acid or 3,3?-dithiodipropionic acid) (500 mg, 2.4 mmol) indry DCM (10 mL)was added EDC?HCI (1.37 g, 7.1 mmol) and N-hydroxysuccinimide (656.8mg, 5.7 mmol). The mixture was stirred for 2 hours at rt. The organic phase was washed witha 2.5% aqueous solution of NaHSO4 (2 x 10 mL) and brine (10 mL), dried over anhydrous Na2504, filtered and concentrated in vacuo. No further purification was performed. Both compounds 1 and 2 were isolated as white solids with full conversion.
...preferably 15 amino acids.When sc(Fv)2 contains three peptide linkers, their length may be all the same or different.For example, such peptide linkers include:Synthetic linkers (chemical crosslinking agents) is routinely used to crosslink peptides, and for example:N-hydroxy succinimide (NHS),disuccinimidyl suberate (DSS),bis(sulfosuccinimidyl) suberate (BS3),dithiobis(succinimidyl propionate) (DSP),dithiobis(sulfosuccinimidyl propionate) (DTSSP),ethylene glycol bis(succinimidyl succinate) (EGS),ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS),disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST),
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃; for 2h;
To the solution of 3,3'-dithioldipropionic acid (1 g, 4.75 mmol) in dichloromethane (20 mL), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC) (2.16 g, 10.5 mmol) and N-hydroxysuccinimide (1.21 g, 10.5 mmol) were added. The mixture was stirred for 2 h at room temperature. The solution was concentrated in vacuo, and the obtained residue was subjected to silica column chromatography (ethyl acetate/hexane = 1:5) to give the NHS ester 2 (1.63 g, 85%). 1H NMR (DMSO-d6, 400 MHz) delta 3.16 (m, 8H), 2.82 (s, 8H). FAB (DMSO-d6) [(M+H)+] calcd 405, found 405.
70%
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃;
One kind of bis(succinimidyl) 3,3'-dithiodipropionate synthesis, the following steps: -; Weigh 3,3'-thiodipropionic acid (6.0g, 0.028mol), N-hydroxysuccinimide (8.0g, 0.06mol), EDC hydrochloride (13g, 0.06 mol), into a 100mL round bottom flask, 40mL of methylene chloride was added, stirred at room temperature, the solid gradually dissolved overnight reaction, there is generated a white solid. Filtered and dried to give the product (8.05g, white solid), yield 70%.
49%
With dicyclohexyl-carbodiimide; In dichloromethane; acetone; at 20℃; for 24h;
. 3,3'-Dithiobis(propanoic acid) (9.5 g, 45.2 mmol) was dissolved in a mixture of acetone (600 mL) and dichloromethane (600 mL) with stirring at room temperature. N-Hydroxysuccinimide (12.68 g, 110.2 mmol, 2.44 eq) was dissolved in the solution, then 1,3-dicyclohexylcarbodiimide (25.9 g, 125.5 mmol, 2.78 eq) was cautiously added. The mixture was allowed to stir at room temperature for 24 hours, after which time the solution was vacuum filtered and the residual solid discarded. The solvent was removed from the filtrate under vacuum, and the oily residue was redissolved in dichloromethane (ca 200 mL). The solution was reduced in volume (ca. 50 mL) and cooled, giving the product as a colourless, crystalline solid (8.90 g, 49% yield). 1H-NMR (d6-DMSO): delta 2.80 (s, 8H), 3.05 (m, 8H). 13C-NMR (d6-DMSO): delta 25.8, 30.7, 32.3, 167.9, 170.4 ppm.
With dicyclohexyl-carbodiimide; In N,N-dimethyl-formamide; for 24h;Cooling with ice;
Under ice-cooling, 4.2504 g (0.02 mol) of <strong>[1119-62-6]3,3'-dithiodipropionic acid</strong> (DTPA)And 4.6036 (0.04 mol) of N-hydroxysuccinimide (HOSu) were dissolved in 20 mL of dimethylformamide;4.1266 g (0.02 mmol) of dicyclohexylcarbodiimide was dissolved in 20 mL of dimethylformamide;Under stirring, it was added dropwise to the reaction mixture, the reaction was stirred in an ice bath for 24 hours, filtered;It is then diluted with ethyl acetate and diluted hydrochloric acid is added to remove the remaining dicyclic urea.The solvent is then removed by evaporation to give a solid product which is washed three to five times with dilute hydrochloric acid and then ethyl acetate. The aqueous hydrochloric acid solution is removed and the solvent is removed by rotary evaporation to give the product, DTSP.
N-{4-[(2R,3R)-3-(4-Nitro-phenyl)-aziridin-2-ylmethyl]-phenyl}-3-(2-{4-[(2R,3R)-3-(4-nitro-phenyl)-aziridin-2-ylmethyl]-phenylcarbamoyl}-ethyldisulfanyl)-propionamide[ No CAS ]
disulfide of disodium N-(3-mercaptopropanoyl)-L-aspartate[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
53%
. L-Aspartic acid (0.42 g, 3.16 mmol) was mixed with sodium hydrogen carbonate (0.50 g, 5.95 mmol, 1.9 eq), then water (1 mL) was added. The sodium hydrogen carbonate was added to quench the acidic hydrogens present in L-aspartic acid. When evolution of carbon dioxide had subsided, the mixture was diluted with 0.5 M bicarbonate buffer, pH 9 (19 mL, 9.5 mmol). All solids dissolved with swirling and the solution was found to be pH 9. 3,3'-Dithiobis(propanoic acid, succinimidyl ester) (0.247 M in DMSO, 5 mL, 1.24 mmol) was added dropwise to the aqueous carbonate solution with strirring. Immediately, a white precipitate appeared, but upon vigorous shaking it redissolved. After 24 hours, during which time the mixture was periodically shaken, dichloromethane (1 mL) was added, and the mixture was again allowed to stand for 24 hours with periodic shaking. The solution, (pH 9) was acidified to pH 7 by the dropwise addition of 32% hydrochloric acid. The product was precipitated by the slow, dropwise addition of the solution to a stirred beaker of absolute ethanol (300 mL) at room temperature, giving a fluffy white precipitate which was collected by filtration and dried under vacuum, giving the product as a white solid (0.37 g, 53% yield). 13C-NMR (D2O): delta 33.2, 34.8, 37.2, 50.9, 175.6, 176.0, 176.4 ppm.
With m-chloroperoxybenzoic acid; In dichloromethane; chloroform; ethyl acetate; acetone;
In step 2 Dithio-bis-(succinimidyl propionate)-S,S-dioxide (DTSPO) was prepared by adding 0.91 g 85% m-chloroperoxybenzoic acid (in 50 ml chloroform) dropwise over an hour into a solution of 0.97 g dithio-bis-(succinimidyl propionate) (DTSP) in 350 ml chloroform. The reaction was stirred for 4 hrs. on ice, then 1 hr. at room temperature. The solvent was removed by rotary evaporation, and the residue washed with benzene. The crude product was extracted with 60 ml dioxane and precipitated with a minimum amount of hexane. Yield: 0.40 g (40%). The product migrated as a single spot on thin layer chromatography in two solvent systems. Rf in methylene chloride:acetone:formic acid 100:25:1=0.30; Rf in ethylacetate=0.50. IR peaks characteristic of the succinimide ester appeared at 1810, 1780 and 1680 cm-1.
In dichloromethane; water; dimethyl sulfoxide; N,N-dimethyl-formamide; at 20℃; for 3h;Product distribution / selectivity;
To a solution of about 0.25 gm (1 mmole) of primaquine in 12.5 mL of about 60% DMF and 12% DMSO in water, was added about 0.35 gm (0.9 mmoles) of DTSP in 6 mL of about 16% CH2Cl2 in DMF. The solution of PQ-DTSP was mixed and put in the dark at rt for about 3 hours before preparing biocleavable conjugates with the following combinative agents.; Preparation X-A Biocleavable <strong>[90-34-6]Primaquine</strong>-Gamma Globulin Conjugate In this example (N45B), primaquine is coupled to gamma globulin protein to provide a biocleavable primaquine protein carrier. Nucleic acid, CCA or other active agent can then be coupled to the gamma globulin. To a solution of about 0.25 gm (1 mmole) of primaquine in 12.5 mL of about 60% DMF and 12% DMSO in water, was added about 0.35 gm (0.9 mmoles) of DTSP in 6 mL of about 16% CH2Cl2 in DMF. The solution of PQ-DTSP was mixed and put in the dark at rt for about 3 hours before preparing a biocleavable conjugate with the gamma globulin.; To a solution of about 0.25 gm (1 mmole) of primaquine in 12.5 mL of about 60% DMF and 12% DMSO in water, was added about 0.35 gm (0.9 mmoles) of DTSP in 6 mL of about 16% CH2Cl2 in DMF. The solution of PQ-DTSP was mixed and put in the dark at rt for about 3 hours before preparing a biocleavable conjugate with the HSA.; In this example (N45), primaquine is coupled to a polylysine peptide to provide a primaquine-peptide carrier. To a solution of about 0.25 gm (1 mmole) of primaquine in 12.5 mL of about 60% DMF and 12% DMSO in water, was added about 0.35 gm (0.9 mmoles) of DTSP in 6 mL of about 16% CH2Cl2 in DMF. The solution of PQ-DTSP was mixed and put in the dark at rt for about 3 hours before preparing a biocleavable conjugate with the gamma globulin.
With triethylamine; In N,N-dimethyl-formamide; at 20℃; for 16h;
For the preparation of the macromolecular derivatives of the phallotoxins, namely phalloidin and phallacidin, 2.0 mmol of toluene-4-sulfonic chloride was dissolved in 400 ul chloroform and added to 0.127 mmol of phalloidin or phallacidin dissolved in 1 ml ice cold pyridine, drop by drop under stirring. After 30 min the reaction was stopped by the addition of 20 ml diethylether. The precipitate was washed with another 20 ml of diethylether and separated by chromatography on Sephadex LH20 with an overall yield of 68 %. For reaction with ammonia, 0.42 mmol monotosyl-phalloidin or monotosyl-phallacadin were reacted with 40 ml ice cold ammonia in methanol (2.5 N), and evaporated after 120 min. Yield of amino-phalloidin and amino-phallacidin was ca. 90%. For coupling to macromolecules the toxin derivatives bearing the functional amino group were reacted with the homobifunctional cross-linking reagent dithiobis-(succinimidylpropionate, DSP). For this, 248 mumoles DSP were dissolved in 1.0 ml dimethylformamide and added to 63 mumoles of dried aminophalloidin. Reaction was started with 2 mul of triethylamine and proceeded under magnetic stirring for 16h at RT. The reaction product was precipitated with 10 ml diethylether and centrifugated, the sediment dissolved in 5 ml methanol and developed on a Sephadex-LH20 column using methanol. Yield of DSP-phalloidin or DSP-phallacidin was about 80 %, purity was minimum 90 %. For reaction with the macromolecules, 7 mg DSP-phalloidin was dissolved in 0.5 ml dimethylformamide and added to the macromolecule dissolved in 0.5 ml PBS. After reaction for 16h at RT, high-molecular weight products were separated by gelfiltration chromatography on Sephadex G-25 using 0.1% NaCl as solvent. After lyophilisation, yield was determined by measuring the characteristic absorption of phalloidin and phallacidin at 300 nm (epsilon=10100). We calculated that with polylysine, for example, one out of 10 lysine residues was conjugated with aminophalloidin or aminophallacidin.
For activation of Pluronic block copolymer, 330 mg of Pluronic amine was first dissolved in 2.5 mL of methanol and added dropwise to a stirring solution of 80 mg of dithiobis(succinimidyl) propionate (DSP) in 2.5 mL of DMF at 25 C. Following the 30 min reaction, the DSP modified Pluronic was isolated by gel filtration on Sephadex LH 20 column (2.5×30 cm) in methanol (fractions 8 mL/4 min). TLC: Rf 0.5-0.6 (Merck Silicagel plates, dichloromethane-methanol, 9:1). The product developed yellow color after exposure of TLC plates to iodine vapors (test on the presence of Pluronic polymers). Polymer-containing fractions were collected and concentrated in vacuo. The N-hydroxysuccinimidocarboxyl (NHS) product (Pluronic-NHS) was dissolved in 1.5 mL of 20% ethanol immediately before reaction with protein
6A,6D-diamino-6A,6D-deoxy-β-cyclodextrin bis(hydrogen carbonate)[ No CAS ]
poly[6A,6D-diamino-6A,6D-dideoxy-β-cyclodextrin bis(hydrogen carbonate)-co-dithiobis(succinimidyl propionate)][ No CAS ]
Yield
Reaction Conditions
Operation in experiment
85%
With sodium hydroxide; In chloroform; water; for 0.5h;pH 10;
A 20 mL scintillation vial was charged with a solution of 92.6 mg (7.65 x 10-5 mol) of the bis(hydrogen carbonate) salt of 4 in 1 mL of water. The pH of the solution was adjusted to 10 with 1 M NaOH before a solution of 30.9 mg (7.65 x 10-5 mol) of dithiobis(succinimidyl propionate) (DSP, Pierce Chemical Co. of Rockford, IL) in 1 mL of chloroform was added. The resulting biphasic mixture was agitated with a Vortex mixer for 0.5 h. The aqueous layer was then decanted and extracted with 3 x 1 mL of fresh chloroform. The aqueous polymer solution was then subjected to gel permeation chromatography (GPC) on Toyopearl HW-40F resin using water as eluant. Fractions were analyzed by GPC and appropriate fractions were lyophilized to yield 85 mg (85%) as a colorless amorphous powder.
2A,3A-diamino-2A,3A-dideoxy-β-cyclodextrin[ No CAS ]
[ 57757-57-0 ]
poly[2A,3A-diamino-2A,3A-dideoxy-β-cyclodextrin-co-disuccinimidyl suberate][ No CAS ]
Yield
Reaction Conditions
Operation in experiment
With sodium hydroxide; In chloroform; water; for 0.5h;pH 10;
A 8 mL vial was charged with a solution of 102.3 mg (8.80 x 10-5 mol) of 2A,3A-diamino-2A,3A-dideoxy-beta-cyclodextrin in 1 mL of water. The pH of the solution was adjusted to 10 with 1 M NaOH before a solution of 36.4 mg (8.80 x 10-5 mol) of dithiobis(succinimidyl propionate) (DSP, Pierce Chemical Co. of Rockford, IL) in 1 mL of chloroform was added. The resulting biphasic mixture was agitated with a Vortex mixer for 0.5 h. The aqueous layer was then decanted and extracted with 3 x 1 mL of fresh chloroform. The aqueous polymer solution was then subjected to gel permeation chromatography.
Tyramine (0.24g, 1.75mmol) was added to a solution of DIC (0.12g, 0.35mmol) in isopropanol (15mL). The reaction mixture was stirred for 24 h at 40C. The mixture was concentrated and purified by column chromatography (EtOAc) to give tyramine- DIC (0.1 Og, 99%) as light yellow solid. Tyramine (0.24g, 1.75mmol) was added to a solution of DSP (0.14g, 0.35mmol) in DMF (4mL). The reaction mixture was stirred for 24 h at 40C. The reaction was stopped by adding water (15 mL) to the reaction mixture. Then the product was filtered and washed with water (10 mL) three times. The product tyramine-DSP (light yellow solid) was then dried and weighed (0.1 lg). The products tyramine-DSP and tyramine -DIC were added to dithiothreitol solution (50 mM, PBS) at 37 C and stirred for 24 h . Then the solutions were lyophilized. Isopropanol (1 mL) was added to the powder acquired and bath sonicated for 15 min. The supernatants from the solutions were collected and analyzed by gas chromatography-mass spectrometry (Alilent Technologies 5975 series MSD, 7820A GC system) and untreated tyramine was used as standard.Tyramine generated from tyramine-DIC was purified through thin layer chromatography (TLC) (EtOAc 90%, methanol 10%). 1H NMR (bruker Avance 400WB) and mass spectrometry (Agilent technologies 6210 LC-TOF) were used to confirm the structure of the compound.
With N-ethyl-N,N-diisopropylamine; In dimethyl sulfoxide; at 20℃; for 5h;
Ac-IR7 (0.3 mg, 355 mumol) was dissolved in DMSO (275 muL) and 14 muL of a 1mMDSP solution was added (1 eq.) along with diisopropylethylamine (0.2 muL, 1 eq.). The mixture was incubated at room temperature for 5 h with periodic mixing, before being concentrated to dryness using a SpeedVac concentrator. The residue was dissolved in water and desalted by ZipTip (Millipore, Bedford, MA, USA) before MS analysis. These non-physiological experimental conditions were employed to attain high yields of the cross-linked products for analysis, allowing for characterization of the fragmentation pathways with relative ease from this model system.
N-(6-aminohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide[ No CAS ]
N-(6-[3-({3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropyl}disulphanyl)propanoyl]amino}hexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
68%
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 2h;
8 mg (10 mumol) of Intermediate 101 were dissolved in 2 ml of DMF and admixed with 8.6 mg (20 mumol) of 1,1?-{-disulphanediylbis[(1-oxopropane-3,1-diyl)oxy]dipyrrolidine-2,5-dione and 3.7 mul of N,N-diisopropylethylamine. The reaction mixture was stirred at RT for 2 h and then the solvent was evaporated off under reduced pressure and the residue was purified by preparative HPLC. 7.2 mg (68% of theory) of the title compound were obtained. [2313] HPLC (Method 5): Rt=1.9 min; [2314] LC-MS (Method 11): Rt=0.94 min; MS (ESIpos): m/z=615 [ (M+2H+]
68%
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 2h;
[4692] 8 mg (10 f.tmol) oflntermediate 101 were dissolvedin 2 ml of DMF and admixed with 8.6 mg (20 f.tmol) of1, 1 '-{ disulphanediy Ibis [ ( 1-oxopropane-3, 1-diyl )oxy]dipyrrolidine-2,5-dione and 3.7 fll ofN,N-diisopropylethylamine.The reaction mixture was stirred at RT for 2 h, andthen the solvent was evaporated off in vacuo, and the residuewas purified by means of preparative HPLC. 7.2 mg (68% oftheory) of the title compound were obtained.[4693] HPLC (Method 5): R,=l.9 min;[4694] LC-MS (Method 11): R,=0.94 min; MS (ESipos):m/z=615 [I/2 (M+2W]
With N-ethyl-N,N-diisopropylamine; In dimethyl sulfoxide; at 20℃; for 24h;
Detailed description of the synthesis and characterization of TPETP-NH2 can be found in the Supplementary Methods. Amine terminated DEVD-TPS-cRGD (10.0 mg, 6.0 mupiiotaomicron) and TPETP-NH2 (3.6 mg, 6.0 muetaiotaomicron) were dissolved in anhydrous DMSO (1.0 mL) in the presence of DIPEA (1.0 mu). The mixture was stirred for 10 min at room temperature. Then dithiobis(succinimidyl propionate) (DSP, 2.4 mg, 6.0 muiotaetaomicron) in DMSO (0.5 mL) was added quickly to the above solution. The reaction was continued with stirring at room temperature for another 24 h. The final product was obtained after purification using preparative HPLC and lyophilized under vacuum to yield the probe TPETP-SS-DEVD-TPS-cRGD as yellow powders in 32% yield (4.7 mg). HPLC (lambda = 320 nm): purity 97.3%, retention time 12.3 minutes; ESI-MS: m/z [M+2H]2+ calc. 1216.945, found 1215.916.
The synthetic route for compound 94 is outlined in Figure 3. (0810) DTT method: Compound 29 (5 mg, 0.003 mmol) was dissolved in pH 7.4 PBS buffer (5 mL) and DSP (dithiobis(succinimidyl propionate)) (20 mg, 0.049 mmol) in DMSO (2.5 mL) was added at room temperature overnight. After that, dithiothreitol (DTT) (4 mg, 0.025 mmol) was added and the solution stirred at 40 C for further 2 h. Compound 91 was purified through Sephadex G-25 column chromatography (4.5 mg, 88%). HRMS (ESI+) Calcd for C5iH99NO46P S [M+2Na+2K-4H]" 1736.2793, found 1736.279.
(S)-22-(4-(((2-amino-4-hydroxypteridin-6-yl)methyl)amino)benzamido)-1-(6-(((di(pyridin-2-yl)methyl)(pyridin-2-ylmethyl)amino)methyl)pyridin-3-yl)-1,7,14,19-tetraoxo-10,11-dithia-2,6,15,18-tetraazatricosan-23-oic acid[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
29%
In dimethyl sulfoxide; at 20℃;
General procedure: 94 mg (0.2 mmol) N4Py-propylamine (1) and 48 mg (0.1 mmol) 2-Aminoethylfolic acid gamma-amide (2) were dissolved in 10 mL DMSO with assistance of mild heating and sonication. A catalytic amount of triethylamine (0.04 mL) and 74 mg (0.2 mmol) DSS (3) were added successively and the mixture was stirred at r.t. overnight. The mixture was then filtered to remove any trace of solid residue and the filtrate was slowly added to vigorously stirred diethyl ether (100 mL). After standing for 1 minute, the ether was decantedcarefullyand the remaining sticky yellow solid was treated with another 100 mL diethyl ether and vigorous stirring. After decanting the ether, the sticky yellow solid was washed with 2x50 mL MeOH and 1x50 mL diethyl ether. After each washing step the suspension was homogenized using a sonication bath and the solid was collected by centrifugation (5 min, at 4000 rpm). 34 mg (0.03 mmol, 31%) of a bright yellow solid was obtained. For cell studies, an analytically pure sample was obtained by preparative RP-HPLC (linear gradient 90% B to 30% B in 40 minutes, flow: 1.0 mL/min, solvent A: acetonitrile B: ammonium formate 5 mM pH = 8.5). Analytical rp-HPLC (linear gradient 90% B to 30% B in 40 minutes, flow: 0.5 mL/min, solvent A: acetonitrile B: ammonium formate 5mM pH=8.5) TR= 24.7 min. LC-MS (ESI+) m/z: 1089.5 [M+H]+, 1111.5 [M+Na]+, 795.4 [M - Pteroic acid + 2H]+, 545.3 [M+2H]2+, 363.8 [M+3H]3+, 295.1 [Pteroic acid]+. HRMS (ESI+) calcd for C56H65N16O8+[M+H]+: 1089.517, found 1089.516.
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