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[ CAS No. 116-31-4 ] {[proInfo.proName]}

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Chemical Structure| 116-31-4
Chemical Structure| 116-31-4
Structure of 116-31-4 * Storage: {[proInfo.prStorage]}
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Product Details of [ 116-31-4 ]

CAS No. :116-31-4 MDL No. :MFCD00001550
Formula : C20H28O Boiling Point : -
Linear Structure Formula :- InChI Key :NCYCYZXNIZJOKI-OVSJKPMPSA-N
M.W : 284.44 Pubchem ID :638015
Synonyms :
trans-Retinal;Retinaldehyde;NSC 626581;NSC 122757;Retinal;Retinene;Vitamin A aldehyde

Calculated chemistry of [ 116-31-4 ]

Physicochemical Properties

Num. heavy atoms : 21
Num. arom. heavy atoms : 0
Fraction Csp3 : 0.45
Num. rotatable bonds : 5
Num. H-bond acceptors : 1.0
Num. H-bond donors : 0.0
Molar Refractivity : 93.71
TPSA : 17.07 Ų

Pharmacokinetics

GI absorption : High
BBB permeant : No
P-gp substrate : No
CYP1A2 inhibitor : Yes
CYP2C19 inhibitor : No
CYP2C9 inhibitor : Yes
CYP2D6 inhibitor : No
CYP3A4 inhibitor : No
Log Kp (skin permeation) : -3.6 cm/s

Lipophilicity

Log Po/w (iLOGP) : 3.96
Log Po/w (XLOGP3) : 6.24
Log Po/w (WLOGP) : 5.72
Log Po/w (MLOGP) : 4.39
Log Po/w (SILICOS-IT) : 5.95
Consensus Log Po/w : 5.25

Druglikeness

Lipinski : 1.0
Ghose : None
Veber : 0.0
Egan : 0.0
Muegge : 2.0
Bioavailability Score : 0.55

Water Solubility

Log S (ESOL) : -5.2
Solubility : 0.00178 mg/ml ; 0.00000624 mol/l
Class : Moderately soluble
Log S (Ali) : -6.38
Solubility : 0.000117 mg/ml ; 0.000000412 mol/l
Class : Poorly soluble
Log S (SILICOS-IT) : -3.75
Solubility : 0.051 mg/ml ; 0.000179 mol/l
Class : Soluble

Medicinal Chemistry

PAINS : 0.0 alert
Brenk : 3.0 alert
Leadlikeness : 1.0
Synthetic accessibility : 4.16

Safety of [ 116-31-4 ]

Signal Word:Warning Class:N/A
Precautionary Statements:P280 UN#:N/A
Hazard Statements:H302-H315 Packing Group:N/A
GHS Pictogram:

Application In Synthesis of [ 116-31-4 ]

* 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.

  • Downstream synthetic route of [ 116-31-4 ]

[ 116-31-4 ] Synthesis Path-Downstream   1~88

  • 1
  • [ 107-86-8 ]
  • [ 3917-41-7 ]
  • [ 116-31-4 ]
YieldReaction ConditionsOperation in experiment
95% In water at 20℃; for 0.0833333h; 13 Step 13 1) To a solution of 0.31 g of 3-methyl-2-butenal in 8 mL of water, quickly add 0.12 g of β-C15 aldehyde and stir at room temperature. 2) After 5 minutes, filter the resulting solid and dry it. 0.39 g (yield 95%) of all trans retinal was obtained. This stage is characterized by Knoevenagel condensation and all trans retinal formation.
With piperidine; ethanol; acetic acid at 15 - 20℃;
  • 2
  • [ 116-31-4 ]
  • [ 1126-58-5 ]
  • [ 121974-71-8 ]
  • 3
  • [ 116-31-4 ]
  • [ 68-26-8 ]
YieldReaction ConditionsOperation in experiment
With diisobutylaluminium hydride
With triethylaluminum
With sodium tetrahydroborate In ethanol Ambient temperature; 2-3 h;
With sodium tetrahydroborate In ethanol at 0℃; All-trans-retml was obtained by reduction of all- trans-retmal with an excess of NaBH4 in EtOH at O0C and purified by normal phase HPLC (Beckman Ultrasphere Si 5μ 4.5x250 mm, 10% EtOAc/hexane; detection at 325 nm). Purified EPO an-rrans-retinoi was αrieα unαer a stream of argon and dissolved in DMF to a final concentration of 3 mM and stored at -8O0C. Retinoid concentrations in EtOH were determined spectrophotometrically. Absorption coefficients for Ret-NH2s were assumed to be equal to those of retinol isomers. Hubbard, et al, Methods in Enzymology 18: 615-653, 1971; Robeson, et al, J. Am Chem Soc 77: 4111-4119.
With sodium tetrahydroborate In ethanol at 0℃; 1 All-trans-retinol is obtained by reduction of all- trans- retinal with an excess OfNaBH4 in EtOH at 00C and purified by normal phase HPLC (Beckman Ultrasphere Si 5μ 4.5x250 mm, 10% EtOAc/hexane; detection at 325 nm). Purified all-fr-αns-retinol is dried under a stream of argon and dissolved in DMF to a final concentration of 3 mM and stored at -800C. Retinoid concentrations in EtOH are determined spectrophotometrically. Absorption coefficients for Ret-NH2s (retinylamines) are assumed to be equal to those of retinol isomers (Hubbard et al., Methods Enzymol. 18:615-53 (1971); Robeson et al., J. Am. Chem. Soc. 77:4111-19 (1955)).
With potassium phosphate; recombinant rat brain aldo-keto reductase R1B10; NADP In methanol Enzymatic reaction;
With potassium dihydrogenphosphate; human N-terminally His10-tagged aldo-keto reductase 1B10; potassium chloride; NADPH at 37℃; Enzymatic reaction;
With rabbit 3-hydroxyhexobarbital dehydrogenase (AKR1C29); NADPH In aq. phosphate buffer; ethyl acetate at 37℃; for 0.5h; Enzymatic reaction; 2.6 Product identification General procedure: The reaction was conducted at 37°C in a 2.0-mL reaction mixture, containing coenzyme (1-mM NADP+ or 0.1-mM NADPH), substrate (0.05-0.1mM), enzyme (0.1-0.3mg), and 0.1-M potassium phosphate, pH 7.4. The substrate and products were extracted into 4-mL ethyl acetate 30min after the reaction was started at 37°C. The products of oxidoreduction of steroids [25] and reduction of PGD2 [28], farnesal [29] and 4-oxo-2-nonenal [18] were analyzed by TLC, as described. The reduced products of TBE were identified by the HPLC methods [23]. The products of 3HB oxidation, 3OB reduction, 5β-androstane-3α,17β-diol oxidation and 5β-androstan-3α-ol-17-one reduction were analyzed by the liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) using a Hewlett-Packard HP 1100 Series LC/MSD system attached with a diode array detector and a column (Mightysil RP-18 GP 5μm, 4.6mm×250mm, Kanto Chemical Co., Tokyo, Japan). Separations were carried out at a flow rate of 0.5mL/min and 40°C using the following mobile phases: 25% acetonitrile aqueous solution containing 0.1% formic acid for 3OB and α/β-3HBs, and 80% acetonitrile aqueous solution containing 0.1% formic acid for the two steroids. 3OB, α-3HB, β-3HB, 5β-androstan-3α-ol-17-one and 5β-androstane-3α,17β-diol were detected by monitoring their total ions (m/z 249.1, 251.1, 251.1, 289.4 and 291.4, respectively) in the negative ESI mode, and eluted at the retention times of 20.1, 17.6, 16.8, 14.9 and 12.7min, respectively. The detection limits of 3OB, α/β-3HBs and the two steroids were 0.1, 0.1 and 1nmol, respectively.

  • 4
  • [ 7235-40-7 ]
  • [ 116-31-4 ]
YieldReaction ConditionsOperation in experiment
89% With oxygen; In water; under 760.051 Torr; for 8h; In a 500 ml reaction bottle, 200 mL of solvent water, 54 g (0.1 mol) of <strong>[7235-40-7]beta-carotene</strong>, and 0.24 g (0.001 mol) of catalyst were added in sequence. Oxygen was maintained and the pressure was maintained at 1 atmosphere. After 8 hours of reaction under sunlight It was extracted three times with water and ethyl acetate. The aqueous layer was removed and the organic layer was dried over anhydrous sodium sulfate. The solvent was removed on a rotary evaporator to obtain 50.5 g of retinal with a yield of 89%.
  • 5
  • [ 68-26-8 ]
  • [ 116-31-4 ]
YieldReaction ConditionsOperation in experiment
90% With manganese(IV) oxide; Retinol (C20H30O, 286.45 g/mol) can be converted to retinal (C20H28O, 284.44 g/mol) according to the following protocol:
29% With manganese(IV) oxide; sodium carbonate; In tetrahydrofuran; at 0℃; for 1h; The retinol (2.1 g, 7.34 mmol) and Na2CO3(11.66 g, 0.11 mol)was dissolved in 130mL THF and cooled to 0 C inice bar. The MnO2 (9.57 g, 0.11 mol) was slowly added into abovecooling mixture. The reaction mixture was continually stirred for1 h after removing the ice bar. The reaction residue was separatedby vacuum filtration, retaining filtrate [27]. The crud product wasconcentrated and further purified by silica gel column chromatography,obtaining light yellowwaxy solid 0.6 g, yield 29%.1H NMR(400 MHz, CDCl3) d 10.11 (d, J 8.2 Hz, 1H), 7.14 (dd, J 15.1,11.5 Hz,1H), 6.36 (dd, J 15.5, 11.5 Hz, 2H), 6.22e6.13 (m, 2H), 5.97 (d,J 8.2 Hz, 1H), 2.33 (d, J 0.9 Hz, 3H), 2.07e1.98 (m, 5H), 1.72 (s,3H), 1.65e1.60 (m, 3H), 1.49e1.45 (m, 2H), 1.04 (s, 6H).
With manganese(IV) oxide; Figure 5 shows synthesis and HPLC separation of retinylamine isomers. (A) Ret- NH2 was synthesized by oxidation of retinol to retinal with MnO2 (shift of Alambdamax from 325 to 383 nm). The oxidation product was further reacted with NH3 in order to produce Ret- NH2 (progress of the reaction was concomitant with blue shift of the absorbance maximum as well as significant red shift upon acidification). Retinylimine was reduced by NABH4 to Ret-NH2 (Alambdamax = 325 nm). Panel B represents the HPLC chromatogram of the isomers' separation (0.5 % NH2in MeOH/EtOAc). The peaks were identified based on their absorbance maxima and shape of the spectra as follows: 1, 11-cw-; 2, 13-cis-; 9- cis-; all-tralphan,s-Ret-NH2. Panel C shows the MS EPO <DP n="37"/>iragmentauon pauem upsiloni cui-iruns-Ret- NH2 with the parent ion at 185 m/z and characteristic retinoid peaks at 268 and 255 m/z.
With manganese(IV) oxide; In ethyl acetate; at 20℃; for 5h;Darkness; Inert atmosphere; Vitamin A-acetate 9 oily concentrate (approx. 0.327 g all-trans retinyl acetate/g oil) (40.00 g, 39.82 mmol) was melted into a round-bottomed flask, and was suspended in 90% (v/v) aqueous ethanol (150 mL) containing sodium hydroxide (5.00 g, 125.00 mmol). The yellow mixture was refluxed for 25 min to achieve saponification to all-trans retinol 10. The clear yellow solution was cooled at 5 C for 30 min, and was mixed with glacial acetic acid (7.2 mL, 125.90 mmol) resulting in pH 5-7. This solution was diluted with water (200 mL), and was extracted with ethyl acetate (EtOAc, 200 mL). The isolated EtOAc phase was washed with water (200 mL). Black, precipitated (active) manganese dioxide powder (63.00 g, MnO2 content ~90%) was suspended in the isolated EtOAc phase, which contained mainly 10, and the mixture was stirred at a temperature of 20 C under light and air exclusion for 5 h. The suspension was occasionally shaken. Afterwards the black suspension was filtered through three layers of filter paper, and the black filter residue was rinsed with EtOAc (150 mL). To remove the finely suspended MnO2 residues, the filtrate, transferred into a separation funnel, was mixed with a freshly prepared solution of l-ascorbic acid (10.00 g, 56.78 mmol) and sodium hydroxide (4.54 g, 113.50 mmol) in water (200 mL). The mixture was shaken vigorously for 1 min. A heavy precipitate of manganese(II) compounds evolved in the aqueous phase, which was dissolved by addition of glacial acetic acid (6.5 mL, 113.66 mmol) and repeated shaking. Finally, the separated EtOAc phase was washed twice with water (2 × 200 mL). The EtOAc phase, containing mainly all-trans retinal 11, was mixed with a solution of thiosemicarbazide (5.73 g, 62.88 mmol) in 90% (v/v) aqueous ethanol (220 mL, prepared by short refluxing). The solution was refluxed for 20 min. The clear, deep yellow solution was then frozen at -25 C for 4 h. After adding 100 ml of water, the volume of the cloudy yellow solution was reduced in vacuo until heavy crystallization started. The crystallizing suspension was additionally frozen at -25 C for 3 h. The evolved orange-yellow crystals were filtered and dried. The orange-yellow filtrate was evaporated to a volume of ca. 150 mL, until it crystallized again. For completion of crystallization, it was frozen at -25 C for 1 h. The evolved orange, fine crystalline precipitate of all-trans retinal thiosemicarbazone was filtered and dried. All crude products were combined (12.00 g) and dissolved in acetone (120 mL) by gentle warming. After 1 h freezing at -25 C water (60 mL) was added, and the crystallizing suspension was frozen at -25 C for 4 h. The evolved orange-yellow crystals were filtered and dried (10.50 g). This material was dissolved in acetone (130 mL) by gentle warming. A solution of sodium hydroxide (1.76 g, 44.00 mmol) in water (7 mL) was added under stirring. Immediately a lemon yellow precipitate of crude compound 12 formed. The precipitating suspension was cooled at 5 C for 2 h. The evolved lemon yellow precipitate of the crude 12 was filtered and dried. The crude product 12 (11.40 g, 66%) was suspended in 80% (v/v) aqueous acetone (600 mL). The suspension was refluxed for 15 min. Afterwards, the orange solution was cooled at 5 C for 5 h, and then was frozen at -25 C for 6 h. The evolved yellow-orange crystalline material (fine needles and accompanying shiny plates) (9.58 g) was filtered and dried. A part of this material (2.00 g) was dissolved in acetone (70 mL) by gentle warming. A solution of sodium hydroxide (0.25 g, 6.25 mmol) in water (1 mL) was added under stirring. Immediately a lemon yellow precipitate formed. The precipitating suspension was frozen at -25 C for 2 h. The evolved lemon yellow precipitate of 12 (2.13 g, 99% for this procedure) was filtered and dried over CaCl2 in vacuo.
With NAD; NADP; 2-amino-2-hydroxymethyl-1,3-propanediol; bovine serum albumin;equine liver alcohol dehydrogenases (ADH) (EC 1.1.1.1 [EC] ); In N,N-dimethyl-formamide; at 37℃; for 1h;pH 8.8;Enzymatic reaction; FIG. 12 shows the oxidation of all-trans-ROL and all-trans-DROL to the respective aldehyde; FIG. 24 shows the metabolism of all-trans-ROL and all-trans-DROL; Oxidation of All-trans-ROL and All-trans-DROL Using Liver Alcohol Dehydrogenase. Equine liver ADH (EC 1.1.1.1 [EC] ) was obtained from Sigma and dissolved in 50 mM Tris (pH 8.8) to a concentration of 5 units/ml (8.6 mg/ml). NAD and NADP were mixed together (1:1) at a concentration of 10 mM each. A substrate solution, 2 mul of 2 mM stock of all-trans-ROL or all-trans-DROL in N,N-dimethylformamide, was added to a 1.5-ml Eppendorf tube containing 20 mul of 10% bovine serum albumin, 20 mul of ADH, 2 mul of cofactor mixture, and 50 mM Tris (pH 8.8) to a total volume of 200 mul. The solutions were incubated at 37 C. for 60 min, after which 50 mul of 0.8 M NH2OH solution (pH 7.0) was added, followed by addition of 300 mul of methanol, 15 min at room temperature, and extraction with 300 mul of hexane. The organic phase was dried and analyzed by normal phase HPLC as described in the analysis of nonpolar retinoids extracted from tissue samples. As a control for the nonenzymatic reaction, boiled protein (90 C. for 5 min) was used with or without addition of cofactors; FIG. 24 shows the metabolism of all-trans-ROL and all-trans-DROL. RetSat saturates all-trans-ROL to all-trans-DROL, which was previously shown to be esterified by LRAT. Here we present evidence demonstrating that the oxidative metabolism of DROL closely follows that of ROL. Broad spectrum enzymes such as SDR and ADH carry out the reversible oxidation of all-trans-DROL to all-trans-DRAL. RALDH1, -2, -3, and -4 oxidize all-trans-DRAL to all-trans-DRA. Several members of the cytochrome P450 enzymes CYP26A1, -B1, and -C1 oxidize all-trans-DRA to all-trans-4-oxo-DRA, identified in vivo and in vitro. Other oxidized all-trans-DRA metabolites, which are not depicted, could be all-trans-4-hydroxy-DRA, all-trans-5,6-epoxy-DRA, all-trans-5,8-epoxy-DRA, and all-trans-18-hydroxy-DRA. The short-chain metabolite C19-ROL is shown here with its possible chemical structure. Its synthetic pathway may proceed from either all-trans-RA by decarboxylation and/or from all-trans-DRA via alpha-oxidation; Given that all-trans-DRA is detected in vivo as a metabolite of all-trans-DROL, we decided to examine its possible mode of synthesis using reconstituted enzyme systems. To oxidize all-trans-DROL to the corresponding aldehyde all-trans-DRAL, we used ADH purified from horse liver (EC 1.1.1.1 [EC]), which is active toward both primary and secondary alcohols. All-trans-DROL and all-trans-ROL were incubated with purified enzyme and the appropriate cofactors. Following the reaction the samples were treated with NH2OH, extracted into the organic phase, and examined by normal phase HPLC. All-trans-RAL or all-trans-DRAL oximes were identified by comparison with synthetic standards. Moise, et al. J. Biol. Chem. 279:50230-50242, 2004. ADH efficiently carried out the conversion of all-trans-ROL to all-trans-RAL and of all-trans-DROL to all-trans-DRAL in the presence of NAD and NADP cofactors (FIGS. 12, A and B) and not in their absence. The boiled enzyme did not exhibit any activity toward either substrate. Next, photoreceptor-specific RDH (prRDH) and RDH12 were tested for ability to catalyze the oxidation of all-trans-DROL to all-trans-DRAL. Both prRDH and RDH12 were active in converting all-trans-ROL to all-trans-RAL but much less so in converting all-trans-DROL to all-trans-DRAL (results not shown).FIG. 12 shows the oxidation of all-trans-ROL and all-trans-DROL to the respective aldehyde. Purified ADH (Sigma) catalyzed the oxidation of all-trans-DROL to all-trans-DRAL (A) and all-trans-ROL to all-trans-RAL (B) in the presence of NAD and NADP. Control reactions using boiled enzyme were negative and show that the conversion is enzymatic. Retinoids were extracted and analyzed by normal phase HPLC. The products of the reaction were syn- and anti-all-trans-DRAL oximes (A) and syn- and anti-all-trans-RAL oximes (B). The experiment was performed in triplicate and repeated.

  • 6
  • [ 867-13-0 ]
  • [ 116-31-4 ]
  • [ 13979-19-6 ]
YieldReaction ConditionsOperation in experiment
96% Stage #1: diethoxyphosphoryl-acetic acid ethyl ester With sodium hexamethyldisilazane In tetrahydrofuran at 0℃; for 0.25h; Stage #2: all-trans-Retinal In tetrahydrofuran at 0 - 20℃; diastereoselective reaction; General Procedure for the Synthesis of C2-ElongetedConjugate Ester by Horner-Wadsworth-Emmons Reaction(GP1) General procedure: To a stirred solution of triethyl phosphonoacetate (C2-phosphonate, 2.5-5.0 eq.) in dry tetrahydrofuran (THF) (0.2 M/phosphonate) was added a solution of NaN(TMS)2 (2.5-5.0 eq.)or n-BuLi (2.5-5.0 eq.) at 0°C, and the mixture was stirred atthe same temperature for 15 min. To this mixture a solutionof aldehyde (1 eq.) in THF (1 M/aldehyde) was added, and thereaction mixture was stirred at room temperature (r.t.) untilthe reaction was completed. The reaction was quenched with asaturated aqueous solution of NH4Cl, and then extracted withethyl acetate. The combined extracts were washed with brine,dried over Na2SO4, and concentrated in vacuo. The residuewas purified by flash chromatography on silica gel to affordthe one double bond elongated conjugate ester. In the case ofA2 analogs, sodium hydride (NaH) (1.5 eq., 60% suspension inmineral oil) was used as a base instead of NaN(TMS)2.
With sodium hydride 1.) THF, 0 deg C, 1 h, 2.) THF; Yield given. Multistep reaction;
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  • [ 14209-41-7 ]
  • [ 5309-25-1 ]
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  • C25H35NO2 [ No CAS ]
  • 10
  • [ 7381-30-8 ]
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  • [ 105539-21-7 ]
YieldReaction ConditionsOperation in experiment
74% With trimethylsilyl trifluoromethanesulfonate In dichloromethane at -78℃; for 4h;
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  • [ 116-31-4 ]
  • [ 302-79-4 ]
YieldReaction ConditionsOperation in experiment
80% With silver(II) oxide; sodium cyanide In methanol for 18h; Ambient temperature;
80% With sodium cyanide; silver(l) oxide 2 Retinal (C20H28O, 284.44 g/mol) can be converted to retinoic acid (C20H28O2, 300.44 g/mol) according to the following protocol:
With silver(II) oxide In methanol Yield given;
With cytosolic aldehyde dehydrogenase from human liver In phosphate buffer; dimethyl sulfoxide at 25℃; for 1h;
Multi-step reaction with 2 steps 1.1: tetracycline-induced HEKK-mouse retinol saturase (RetSat) cells / Enzymatic reaction 2.1: 2-amino-2-hydroxymethyl-1,3-propanediol; bovine serum albumin / retinal pigment epithelium (RPE) microsomes / 1 h / 37 °C / pH 8.8 / Enzymatic reaction 2.2: pH 4.0
Multi-step reaction with 2 steps 1.1: untransfected HEKK cells / Enzymatic reaction 2.1: 2-amino-2-hydroxymethyl-1,3-propanediol; bovine serum albumin / retinal pigment epithelium (RPE) microsomes / 1 h / 37 °C / pH 8.8 / Enzymatic reaction 2.2: pH 4.0
With sodium pyrophosphate; GLUTATHIONE; retinaldehyde dehydrogenase; nicotinamide adenine dinucleotide In aq. phosphate buffer; ethanol at 37℃; for 0.5h; Enzymatic reaction; 4.6. Enzyme activity assays using retinaldehyde Due to overlap in the absorption spectrum of retinaldehyde andNADH, a fluorescence-based NADH assay (Amplite FluorimetricNADH Assay Kit, AAT Bioquest, Inc; Sunnyvale, CA) was employed tomeasure dehydrogenase activity of purified recombinant RALDH isozymeswith the natural substrate, retinaldehyde. Recombinant proteinwas diluted in dialysis buffer (see above) containing 2mM DTT to avolume of 40 μL and added to amber microfuge tubes containing 50 μLof 2× assay buffer [5% DMSO, 8mM NAD+, 64mM tetrasodium pyrophosphate,pH 8.2, 0.2mM pyrazole, 10mM reduced glutathione(GSH), 1.9mM EDTA] at 25 °C. Retinaldehyde was used directly fromsealed ampules (Sigma, ≥98% purity, 25 mg/vial), stored at -20° anddiscarded after 1 week. Retinaldehyde was initially solubilized inethanol and its concentration determined by absorption spectroscopy(λmax=383 and ε=42,880) of serial dilutions.91 The retinaldehydestock solution was then diluted to 10× concentrations(0.155-2.50 mM) in ethanol. Under dim red light, reactions were initiatedby addition of retinaldehyde in EtOH (10 μL; final retinaldehydeconcentration=15.5-250 μM; final reaction volume=100 μL), andreactions were incubated in a water bath for 30 min at 37 °C. Underthese conditions, retinaldehyde remained in solution in synthesis bufferat all concentrations. Reactions were stopped by immersion in icewater. For NADH quantification, a 50 μL aliquot of each reaction wasadded to wells of solid black 96 well microplates (Greiner Bio One;Kremsmünster, Austria) followed by 50 μL of the NADH reaction mixture(Amplite Fluorimetric NADH Assay Kit, according to manufacturer’sdirections). Total NADH was quantified by fluorescence intensityat Ex/Em=540/590 nm and compared with a standard curve(0.1-30 μM NADH). While an excess of NAD+ was added to the reactions,the amount of NADH generated in the 30 min was within therange of the standard curve. For inhibition assays, recombinant proteinwas pre-incubated with DAR at a final concentration of 0-10 μM in DMSO for 0-80 min at 37 °C prior to addition of assay buffer and retinaldehyde(DMSO final concentration after addition of substrate andsynthesis buffer ≤3.5%). The calculations for KI and kinact were performedas previously described
With potassium permanganate; disodium hydrogenphosphate In methanol; water at 25℃; for 0.5h;

  • 27
  • [ 116-31-4 ]
  • [ 20638-88-4 ]
YieldReaction ConditionsOperation in experiment
78.3% With ammonium heptamolybdate; ammonium hydroxide; dihydrogen peroxide; tetra-(n-butyl)ammonium iodide In tetrahydrofuran at 20℃; for 24h;
78% With ammonium hydroxide; ammonium molybdate(VI) tetrahydrate; dihydrogen peroxide; tetra-(n-butyl)ammonium iodide In tetrahydrofuran at 5℃; for 10h;
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YieldReaction ConditionsOperation in experiment
95% With diisobutylaluminium hydride In hexane; Petroleum ether at -60 - -20℃; for 1h;
26.2% With diisobutylaluminium hydride In toluene at -5 - 0℃; for 1h; Inert atmosphere; Preparation of Compound-7: To a stirred solution of Compound-6 (8.7 g, 30 mmol) in dry toluene (43.5 mL) at -5 °C under nitrogen was added dropwise DIBAL-H (1 M in toluene, 31mL). The reaction mixture was stirred at 0 °C for 1 h. the reaction was slowly quenched with potassium sodium tartrate solution and the resulting mixture was filtered through celite. The filtrate was extracted with chloroform thrice. The combined organic layers were dried over anhydrous Na2SO4 and concentrated. The crude compound was purified by column chromatography on 230-400 mesh silica gel and eluted with 10% DCM/petroleum ether to obtain Compound-7 (2.3 g, 26.2%). MS (m/z): [M + H]+, 285.4.
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  • [ 56085-55-3 ]
  • [ 56085-53-1 ]
  • [ 67737-37-5 ]
  • 37
  • [ 116-31-4 ]
  • [ 504-02-9 ]
  • retinylidene-1,3-cyclohexanedione [ No CAS ]
YieldReaction ConditionsOperation in experiment
89% With piperidine In benzene Ambient temperature;
  • 39
  • [ 74916-02-2 ]
  • [ 514-85-2 ]
  • [ 472-86-6 ]
  • [ 116-31-4 ]
  • [ 23790-80-9 ]
  • 40
  • [ 74916-02-2 ]
  • [ 564-87-4 ]
  • [ 472-86-6 ]
  • [ 116-31-4 ]
  • [ 564-88-5 ]
  • 42
  • [ 151018-50-7 ]
  • [ 1209-68-3 ]
  • [ 514-85-2 ]
  • [ 472-86-6 ]
  • [ 116-31-4 ]
  • 43
  • [ 564-87-4 ]
  • [ 514-85-2 ]
  • [ 472-86-6 ]
  • [ 116-31-4 ]
  • [ 564-88-5 ]
  • 46
  • [ 146609-18-9 ]
  • [ 472-86-6 ]
  • [ 116-31-4 ]
  • 47
  • (2E,6E,8E)-4-Chloro-3,7-dimethyl-9-(2,6,6-trimethyl-cyclohex-1-enyl)-nona-2,6,8-trienal [ No CAS ]
  • [ 116-31-4 ]
YieldReaction ConditionsOperation in experiment
86% With 1,8-diazabicyclo[5.4.0]undec-7-ene In dichloromethane at 50℃; for 2h;
  • 48
  • (5E)-8-cyclogeranylidene-7-hydroxy-3,7-dimethyl-octa-3,5-dien-1-al, diethylacetal [ No CAS ]
  • [ 514-85-2 ]
  • [ 472-86-6 ]
  • [ 116-31-4 ]
  • [ 23790-80-9 ]
  • 50
  • [ 472-86-6 ]
  • [ 514-85-2 ]
  • [ 564-87-4 ]
  • [ 116-31-4 ]
  • 52
  • [ 116-31-4 ]
  • [ 14447-18-8 ]
  • Retinyliden-malonsaeure-benzylester-nitril [ No CAS ]
  • 53
  • [ 116-31-4 ]
  • [ 15666-97-4 ]
  • Retinyliden-malonsaeure-octylester-mononitril [ No CAS ]
  • 54
  • [ 564-88-5 ]
  • petroleum ether-solution [ No CAS ]
  • [ 472-86-6 ]
  • [ 116-31-4 ]
  • 55
  • [ 116-31-4 ]
  • [ 514-85-2 ]
  • [ 564-87-4 ]
  • [ 472-86-6 ]
  • [ 564-88-5 ]
  • 56
  • [ 489-84-9 ]
  • [ 116-31-4 ]
  • (2E,4E,6E,8E)-1-(3-guaiazulenyl)-3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2,4,6,8-nonatetraen-1-ylium hexafluorophosphate [ No CAS ]
YieldReaction ConditionsOperation in experiment
91% With hexafluorophosphoric acid In methanol at -10℃; for 1h;
  • 57
  • C62H97NO34 [ No CAS ]
  • [ 116-31-4 ]
  • [ 29390-67-8 ]
  • 58
  • [ 116-31-4 ]
  • [ 56522-24-8 ]
  • [ 886226-24-0 ]
YieldReaction ConditionsOperation in experiment
90% With lithium bromide In tetrahydrofuran at 20℃; for 20h; Inert atmosphere; 2 Example 2
Tert-butyldimethylsilane protected Vitamin A aldehyde nitrile alcohol (VI) 10 g of the vitamin A aldehyde was dissolved in a dry nitrogen-protected solution of 20 ml of tetrahydrofuran, followed by stirring, to the stirring solution was further added 0.5 g of lithium bromide (LiBr) and 70 g of tert-butyldimethylsilyl cyanide. The reaction mixture was further stirred at room temperature for 20 hours, and 20 ml of water was added to terminate the reaction. The reaction mixture was extracted three times with 30 ml of ethyl acetate. The solvent was evaporated and the crude product was isolated by silica gel chromatography (95: 5 hexane / ethyl acetate) to give 14.3 g of compound (VI) as an orange oil.
78% With triethylamine In dichloromethane for 20h;
78% With triethylamine In dichloromethane for 20h; tert-Butyl-dimethylsilylcyanohydrin of retinal (16).; To a flame-dried flask under argon atmosphere was added retinal (15) (1.03 g, 3.62 mmol) dissolved in dry CH2Cl2 (50 mL). A catalytic amount of Et3N (0.1 mL) was added then tert-butyldimethylsilyl cyanide (1.0 g, 7.08 mmol) dissolved in CH2Cl2 (10 mL) was added by cannulation. The reaction stirred for 20 h after which the solution was concentrated, chromatographed (95:5 hexanes/ethyl acetate), dried (Na2SO4) under argon, and subjected to vacuum overnight to give 1.20 g (78%) of orange oil. UV λmax=329 nm (ε=49462); IR (cm-1) 3042 (w), 2960 (s), 2928 (s), 2850 (s), 2239 (w), 1586 (w), 1472 (m), 1358 (m), 1256 (m), 1105 (s), 963 (s), 832 (s), 775 (m); 1H NMR (DMK-d6) δ0.16 (s, 3H), 0.20 (s, 3H), 0.90 (s, 9H), 1.02 (s, 6H), 1.45-1.48 (m, 2H), 1.58-1.63 (m, 2H), 1.70 (s, 3H), 1.99 (s, 6H), 5.57-5.61 (m, 2H), 6.13-6.23 (m, 3H), 6.38 (d, 1H, J=15.2 Hz), 6.86 (dd, 1H, J=11.3, 15.2 Hz); HRMS (ES) calcd for C27H43NOSi (M+Na) 448.3012, found 448.2982.
  • 59
  • [ 116-31-4 ]
  • [ 29390-67-8 ]
  • C62H97NO34 [ No CAS ]
  • 60
  • [ 20638-88-4 ]
  • [ 472-86-6 ]
  • [ 116-31-4 ]
  • 61
  • [ 116-31-4 ]
  • [ 33532-44-4 ]
YieldReaction ConditionsOperation in experiment
80% With N-Bromosuccinimide; N,N-diethylaniline In dichloromethane; water; acetonitrile at -15℃; for 0.166667h;
Multi-step reaction with 3 steps 1: N-bromosuccinimide / CH2Cl2 / 0.13 h / 0 °C 2: aq. KOAc / acetone / 24 h / 20 °C 3: 80 percent / pyridinium dichromate / CH2Cl2 / 1.5 h / Ambient temperature
Multi-step reaction with 3 steps 1: N-bromosuccinimide / CH2Cl2 / 0.13 h / 0 °C 2: aq. AcOH, KOAc / acetone / 24 h / 20 °C 3: 80 percent / pyridinium dichromate / CH2Cl2 / 1.5 h / Ambient temperature
  • 62
  • aqueous potassium hydroxide [ No CAS ]
  • aqueous sodium chlorite [ No CAS ]
  • [ 513-35-9 ]
  • [ 116-31-4 ]
  • [ 302-79-4 ]
YieldReaction ConditionsOperation in experiment
55.9% With phosphoric acid; In 1,4-dioxane; ethanol; hexane; Example 1 Synthesis of vitamin A acid (all trans form) A 1-liter three-necked flask was charged under an atmosphere of nitrogen with 54.9 g of vitamin A aldehyde (69.1% purity, 133.6 mmoles, ratio of all trans form: 98.5%), 250 g of 2-methyl-2-butene, 100 ml of dioxane and 53.2 g of a 25% aqueous sodium chlorite solution. The mixture was mechanically stirred vigorously and, while the internal temperature was maintained at 5 C., 170 g of a 8.5% aqueous phosphoric acid solution was added thereto dropwise over 1.5 hours. After completion of the addition, the mixture was further stirred vigorously at 5 C. for 40 minutes. After confirming exhaustion of the starting material vitamin A aldehyde by thin layer chromatography, yellow solid that precipitated was filtered through a glass filter and then washed several times with water. The yellow solid was transferred to a 500-ml flask, and 200 ml of a 5% aqueous potassium hydroxide solution and 100 ml of ethanol were added thereto. The mixture was refluxed with heating for 1.5 hours. After cooling, the reaction mixture was transferred to a 1-liter separating funnel. To the mixture 200 ml of hexane was added and the funnel was shaken sufficiently. The bottom layer was separated and acidified with a 10% aqueous sulfuric acid solution. The yellow solid that precipitated was extracted with 500 ml of isopropyl ether and the extract was washed with water until the bottom layer became neutral. The isopropyl ether was distilled off under reduced pressure and 400 ml of ethanol was added to the yellow residue and the mixture was heated. After confirmation of complete dissolution, the solution was gradually cooled with stirring to 0 C. The vitamin A acid crystal that precipitated was filtered through a glass filter and then washed with cold ethanol. The crystal was dried under reduced pressure, to give 22.3 g of vitamin A acid having a purity of at least 99%. The yield was 55.9%. The vitamin A acid thus obtained had a all trans form ratio of 99.5%.
  • 63
  • [ 68-26-8 ]
  • [ 630-19-3 ]
  • [ 75-84-3 ]
  • [ 116-31-4 ]
YieldReaction ConditionsOperation in experiment
87% With aluminum isopropoxide; In water; To the obtained crude vitamin A, 1.45 g (16.7 mmol) of trimethylacetaldehyde and 86 mg (0.42 mmol) of aluminum isopropoxide were added, and agitated at 40-45 C. for 50 min. After the stopping of reaction by the addition of 0.07 ml of water, unreacted trimethylacetaldehyde and neopentyl alcohol formed as a reaction by-product were distilled off (at 50 C., 10 mmHg) to obtain 3.36 g of crude vitamin A aldehyde (purity 61.2%, yield 87%, total trans form ratio 98.4%). The obtained crude vitamin A aldehyde was recrystallized from hexane to obtain 1.45 g of purified vitamin A aldehyde (melting point 60-61 C.).
With aluminum isopropoxide; In water; To the obtained crude vitamin A, 6.08 g (16.7 mmol) of trimethylacetaldehyde and 360 mg (1.75 mmol) of aluminum isopropoxide were added, and the reaction mixture was agitated at 45-50 C. for 45 min. After the stopping of reaction by the addition of 0.15 ml of water, unreacted trimethylacetaldehyde and neopentyl alcohol formed as a reaction by-product were removed by distillation under a reduced pressure (50 C., 10 mmHg) to obtain 11.3 g of crude vitamin A aldehyde. The obtained crude vitamin A aldehyde was purified by column chromatography (eluent: hexane/ethyl acetate=85/15) to obtain 7.68 g of purified vitamin A aldehyde (yield 80%, total trans form ratio 98%, melting point 60-62 C.).
  • 64
  • [ 5497-67-6 ]
  • [ 68-26-8 ]
  • [ 3420-42-6 ]
  • [ 116-31-4 ]
YieldReaction ConditionsOperation in experiment
87% With aluminum isopropoxide; In water; To the obtained crude vitamin A, 1.86 g (16.5 mmol) of 2,2-dimethyl-4-pentenal and 86 mg (0.42 mmol) of aluminum isopropoxide were added, and the reaction mixture was agitated at 35-40 C. for 60 min. After the stopping of reaction by the addition of 0.07 ml of water, unreacted 2,2-dimethyl-4-pentenal and 2,2-dimethyl-4-penten-1-ol formed as a reaction by-product were distilled off (at 60 C., 10 mmHg) to obtain 3.77 g of crude vitamin A aldehyde (purity 55.5%, yield 87%, total trans form ratio 98%). The obtained crude vitamin A aldehyde was recrystallized from hexane to obtain 1.25 g of purified vitamin A aldehyde (melting point 60-61 C.).
  • 65
  • [ 116-31-4 ]
  • [ 15647-89-9 ]
  • [ 7235-40-7 ]
YieldReaction ConditionsOperation in experiment
With sodium; In methanol; ethanol; dichloromethane; EXAMPLE 9 13.6 parts by weight of triphenyl-phosphonium chloride are dissolved in 100 parts by volume of absolute ethanol and, while stirring in a nitrogen atmosphere, simultaneously treated dropwise with a solution of 7 parts by weight of vitamin A aldehyde in 100 parts by volume of absolute ethanol and a solution of 0.6 parts by weight of sodium in 15 parts by volume of absolute methanol. After 2 hours the resulting red precipitate is filtered off, washed with water and dissolved in methylene chloride. The methylene chloride solution is filtered after drying over sodium sulfate and carefully evaporated. By spraying in ethanol there are obtained violet crystals of beta-carotene. After recrystallization from benzene/ethanol, there are obtained 9.4 parts by weight of beta-carotene of melting point 177-179 (abs. max. 453, 480 mmu, E11 = 2510, 2185 in petroleum ether).
  • 66
  • [ 1005452-43-6 ]
  • [ 33603-83-7 ]
  • [ 116-31-4 ]
YieldReaction ConditionsOperation in experiment
12.6 mg With potassium phosphate In tetrahydrofuran; toluene at 23℃; for 5h;
With potassium phosphate In tetrahydrofuran at 20℃; for 3h; 20 Example 20Fully Automated Synthesis of All-trans-Retinal Using Aqueous Deprotection ModuleThe following example, while not specific to a PIDA-based system, describes procedures that, when used with a PIDA-based system, are reasonably expected to achieve similarly effective results. The first deprotection tube was prepared as follows: To a new, fritted 12-g cartridge (Luknova, Mansfield, MA 02048, Part No. FC003012) was added trienyl MIDA boronate (345.2 mg, 1 mmol, 9 equivs). To this was added sodium hydroxide (120.0 mg, 3 mmol, 27 equivs). The cartridge was capped with its female Luer-port screw cap. To this Luer port was attached a 5-mL polypropylene syringe barrel (Henke-Sass, Wolf GmbH, Tuttlingen, Germany, 78532, Part No. A5) from which the plunger had been removed. This first deprotection tube was wrapped with aluminum foil.The second deprotection tube was prepared as follows: To a new, fritted 12-g cartridge (Luknova, Mansfield, MA 02048, Part No. FC003012) was added sodium hydroxide (40.0 mg, 1 mmol, 9 equivs). Sodium hydroxide pellets were shaved down to the correct mass with a clean razor blade and massed quickly to minimize adsorption of atmospheric moisture. The cartridge was capped with its female Luer-port screw cap. To this Luer port was attached a 5-mL polypropylene syringe barrel (Henke-Sass, Wolf GmbH, Tuttlingen, Germany, 78532, Part No. A5) from which the plunger had been removed. This second deprotection tube was wrapped with aluminum foil.The first and second predrying tubes were prepared as follows: To a new, fritted 12-g cartridge (Luknova, Mansfield, MA 02048, Part No. FC003012) was added Celite 545 filter aid (800 mg, not acid- washed, Acros Organics, Product No. 349670025, Lot No.A0287832). To this was added anhydrous magnesium sulfate (2.1 g, ReagentPlus, >99.5%, Sigma-Aldrich, Product No. M7506, Lot No. 080M0246V). These two solids were mixed with a spatula until visibly homogenous. On top of the solid mixture was placed a 5- mL polypropylene syringe plunger (Henke-Sass, Wolf GmbH, Tuttlingen, Germany, 78532, Part No. A5), manually cut to approximately 6.5 cm in length. The cartridge was capped with its female Luer-port screw cap. The Luer port was covered tightly with a small square (approximately 1 cm x 1 cm) of aluminum foil. Each predrying tube was wrapped with aluminum foil.The first and second drying tubes were prepared as follows: To a new, fritted 12-g cartridge (Luknova, Mansfield, MA 02048, Part No. FC003012) was added Celite 545 filter aid (300 mg, not acid-washed, Acros Organics, Product No. 349670025, Lot No. A0287832). To this was added activated molecular sieves (3.6 g, 4A, -325 mesh, Sigma-Aldrich, Product No. 688363, Lot No. MKBF4010V). Molecular sieves were activated at 300 °C, ambient pressure, 24 h, and cooled/stored in a vacuum desiccator under dry argon over Drierite. These two solids were not mixed. On top of the layered solids was placed a 5-mL polypropylene syringe plunger (Henke-Sass, Wolf GmbH, Tuttlingen, Germany, 78532, Part No. A5), manually cut to approximately 5.5 cm in length. The cartridge was capped with its female Luer-port screw cap. Each drying tube was wrapped with aluminum foil.The first and second deoxygenating/concentr citing tubes were prepared as follows: A new, fritted 12-g cartridge (Luknova, Mansfield, MA 02048, Part No. FC003012) was capped with its female Luer-port screw cap. Each deoxygenating/concentrating tube was wrapped with aluminum foil.The first reaction tube was prepared as follows: To a new, fritted 12-g cartridge (Luknova, Mansfield, MA 02048, Part No. FC003012) was added a 4-g frit (Luknova, Mansfield, MA 02048, Part No. FC003004). This frit was secured, concentrically, to the 12-g cartridge frit with 26 G Chromel A wire, pierced through the 12-g frit. To this reaction tube was added, in order, anhydrous potassium phosphate (1.39 g, 3 mmol + 750 mg, 27 equivs + 750 mg, 97%, Alfa Aesar, Product No. L15168, Lot No. L02U015), palladium (II) acetate (1.9 mg, 0.0083 mmol, 2.5 mol%, > 99.9%, Sigma-Aldrich, Product No. 520764, Lot No.1000824996), 2-dicyclohexylphosphino-2',6'-dimethyoxy-l,l '-biphenyl (Sphos, 6.8 mg, 0.017 mmol, 5 mol%, 98%, Strem Chemicals, Product No. 15-1143, Lot No. 18526300), vinyl iodide MIDA boronate (103.0 mg, 0.33 mmol, 3 equivs), and a PTFE-coated rare earth magnetic stir bar. Potassium phosphate was freshly ground in a 100 °C mortar and pestle. The cartridge was capped with its customized female Luer-port screw cap. The customized cap consists of a standard female Luer-port cap with a bent (by approximately 45°), 1.5 inch, 18 G, disposable needle installed through the cap and a small ball of Kimwipe inserted into the Luer port. It is important remove the cored-out polypropylene plug from the inside of the needle after installation. The cap was topped with a fritted 4-g cartridge (Luknova, Mansfield, MA 02048, Part No. FC003004).The precipitation tube was prepared as follows: To a new, fritted 12-g cartridge(Luknova, Mansfield, MA 02048, Part No. FC003012) equipped with a PTFE-coated magnetic stir bar was added Celite 545 filter aid (150 mg, not acid-washed, Acros Organics, Product No. 349670025, Lot No. A0287832) and 3-aminopropyl functionalized silica gel (250 mg, 40-63 μιη, approximately 1 mmol/g NH2, Sigma-Aldrich, Product No. 364258, Lot No. 79096HM). The cartridge was capped with its female Luer-port screw cap. To the cartridge was added hexanes (5 mL, reagent grade) and the resulting suspension was swirled vigorously to mix the solids. The mixed suspension was allowed to settle for approximately 5 seconds and then the solvent was drained by forcing a plug of ambient air through the top of the cartridge by syringe. This process firmly embeds the stir bar in the solids to prevent stirring before the precipitation tube is utilized. This precipitation tube was wrapped with aluminum foil.The silica gel chromatography column was prepared as follows: A silica gel chromatography column was freshly prepared from custom PTFE fittings usingunfunctionalized silica gel. The cartridge was modeled after a 4-g cartridge (Luknova, Mansfield, MA 02048, Part No. FC003004), but was made of PTFE instead of polypropylene. To a clean, fritted column was added silica gel. This was done by vacuum aspiration through the bottom male Luer tip fitting. This process ensured tight, even packing of the silica gel plug. Excess silica gel was removed manually with a spatula and a 4-g frit(Luknova, Mansfield, MA 02048, Part No. FC003004) was placed on top of the silica plug. This doubly-fritted cartridge was capped with its female Luer-port screw cap, using PTFE tape to ensure a tight seal.The second reaction vessel was prepared as follows: To a non- flame-dried 7-mL glass vial equipped with a PTFE-coated magnetic stir bar was added palladium (II) acetate (1.2 mg, 0.0056 mmol, 5 mol%, > 99.9%, Sigma-Aldrich, Product No. 520764, Lot No.1000824996), 2-dicyclohexylphosphino-2',6'-dimethyoxy-l, -biphenyl (Sphos, 4.6 mg, 0.011 mmol, 10 mol%, 98%, Strem Chemicals, Product No. 15-1143, Lot No. 18526300), and anhydrous potassium phosphate (212 mg, 1 mmol, 9 equivs, 97%>, Alfa Aesar, Product No. L15168, Lot No. L02U015). Potassium phosphate was freshly ground in a 100 °C mortar and pestle. This vial was sealed with a PTFE-lined septum screw cap. Through the septum was added a 1.5 inch, 20 G, disposable needle connected to a dry argon gas line. Then, through the septum was added a 1.5 inch, 20 G, disposable needle to act as a vent. The reaction vial was then flushed with dry argon for approximately 7 min. The vent needle and then the argon needle were removed from the septum.The tubes, vessels and columns described above were used as follows: See FIG. 13 for reaction scheme. Both deprotection tubes (wrapped with aluminum foil) were securely installed on the machine. Tubes were installed by placing the tube's male Luer tip into the machine's appropriate female Luer port and were secured with a firm downward force and slight (less that one quarter turn) clockwise rotation. Both predrying tubes (wrapped and topped with aluminum foil) were securely installed on the machine. Both drying tubes (wrapped in aluminum foil) were securely installed on the machine. Each drying tube was connected to the inert gas manifold by attaching a patch line to the machine's gas manifold and the tube's top Luer port. Patch lines are approximately 12-inch lengths of tubing with male Luer-tip fittings on both ends. Both deoxygenating/concentrating tubes (wrapped in aluminum foil) were securely installed on the machine. Each deoxygenating/concentrating tube was connected to the inert gas manifold by attaching a vented patch line to the machine's gas manifold and the tube's top Luer port. Vented patch lines are approximately 12-inch lengths of tubing with a male Luer-tip fitting on the machine-end and a Y- connector (one port connected to the line, one port connected to a male Luer-tip fitting, and one port left open) on the tube-end. The first reaction tube was securely installed on the machine (in a heating block preheated to 40 °C) and connected to the inert gas manifold by attaching the reaction vent line to the tube's top Luer port. The reaction tube was then covered with aluminum foil and set to stirring at 600 rpm. The silica gel column was securely installed on the machine and connected to the purification module by attaching the eluent line to the column's top Luer port. The precipitation tube (wrapped in aluminum foil) was securely installed on the machine and connected to the purification module by placing the eluent line (fixed with a 1.5 inch, 18 G, disposable needle) through the tube's top Luer port).The experiment's pre-assembled code was then loaded and executed to begin the automated sequence. The first aqueous MID A boronate deprotection commenced immediately. After running the first deprotection (rt, 10 min), the machine quenched and worked up the resulting boronic acid solution and then dried, deoxygenated, and concentrated it. The machine then ran the first, slow addition, cross-coupling reaction (40 °C, 8 h total) and purified the resulting coupled product. The machine then ran the second aqueous MID A boronate deprotection (rt, 10 min) and subsequently quenched, worked up, dried, deoxygenated, and concentrated the resulting boronic acid solution.Approximately 5 minutes before the second cross-coupling began, the second reaction vessel was placed in an aluminum block (at room temperature) on a stir plate. An inert gas vent line (fixed with a 1.5 inch, 20 G, disposable needle) was connected, through the septum. The reaction tube was then covered with aluminum foil and set to stirring at 600 rpm. Separately, into a non-flame-dried 1.5-mL glass vial was added the aldehyde (16.6 mg, 0.11 mmol, 1 equiv). The vial was sealed with a septum screw cap and to this was added 100 deoxygenated dry THF from a 100 μ, gas tight, fixed needle, glass syringe. The vial was manually gently agitated to dissolve the aldehyde and then was added to the reaction vial with the same syringe. The remaining residual aldehyde was quantitatively transferred to the reaction vial with 2 x 50 of deoxygenated dry THF using the same syringe. As the machine automatically deoxygenated the reaction addition line (fixed with a 1.5 inch, 22 G, disposable needle), it was connected to the reaction vessel, through the septum. The machine then ran the second, fast addition, cross-coupling reaction (rt, 3 h).At the end of 3 hours, the reaction vial was removed from the machine and the crude reaction mixture was filtered through a 1-cm pad of Celite packed in a pipette. The reaction vial was washed with 3 x 2 mL dry THF and these washings were filtered through the Celite pad. The pad was then washed with 3 x 2 mL dry THF. The resulting clear dark yellow filtrate was concentrated in vacuo (rt, 80 Torr), azeotroped with 3 x 5 mL dichloromethane (rt, 80 Torr), and residual solvent was removed on high vacuum (30 min, 200 mTorr) to afford a dark yellow/orange sticky solid. This crude product was manually purified by silica gel flash chromatography to afford a mixture of all-trans-retinal: 13-czs- retinal in a ratio of 1 :0.55 in a combined total 30% yield.
  • 67
  • C16H25BO2 [ No CAS ]
  • [ 14804-55-8 ]
  • [ 116-31-4 ]
YieldReaction ConditionsOperation in experiment
With potassium phosphate In tetrahydrofuran; toluene at 23℃; for 5h; 16 A solution of the palladium catalyst was prepared as follows: To a 1.5 mL vial equipped with a magnetic stir bar and containing 2-dicyclohexylphosphino-2',6'-dimethoxy-1,1'-biphenyl (4d) (3.6 mg, 0.0088 mmol, 2.0 eq.) was added a solution of Pd(OAc)2 in toluene (0.038 M, 0.115 mL, 0.0044 mmol, 1.0 eq.). The vial was sealed with a PTFE-lined cap and maintained at 65° C. with stirring for 15 min.This catalyst solution was then utilized in the following procedure: To a 4 mL vial equipped with a magnetic stir bar and containing enal 85 (10 mg, 0.067 mmol, 1.0 eq.) was added the boronic acid (corresponding to boronate 84; see above) as a solution in THF (estimated 0.101 M, 1 mL, 0.101 mmol, 1.5 eq.), anhydrous K3PO4 as a finely ground powder (42.6 mg, 0.201 mmol, 3.0 eq.), and the catalyst stock solution described above (0.035 mL, 0.0013 mmol Pd, 2 mol % Pd). The resulting mixture was sealed with a PTFE-lined cap and stirred at 23° C. for 5 h. The reaction was then quenched with the addition of saturated aqueous NaHCO3 (2 mL). The layers were separated and the aqueous layer was extracted with Et2O (3×5 mL). The combined organic layers were dried over Na2SO4 and concentrated in vacuo. The crude material was purified by flash chromatography (hexanes:EtOAc 32:1) to yield all-trans-retinal (49) as a bright yellow solid (12.6 mg, 0.044 mmol, 66%). 1H NMR, 13C NMR, HRMS, and IR analysis of synthetic 49 were fully consistent with the data reported for the isolated natural product.
  • 68
  • [ 564-88-5 ]
  • [ 472-86-6 ]
  • [ 116-31-4 ]
  • 69
  • [ 116-31-4 ]
  • [ 67890-45-3 ]
  • [ 302-79-4 ]
  • [ 67890-46-4 ]
YieldReaction ConditionsOperation in experiment
Stage #1: all-trans-Retinal With NAD; NADP; ATP; 2-amino-2-hydroxymethyl-1,3-propanediol; Sucrose at 37℃; for 2h; Enzymatic reaction; Stage #2: With hydroxylamine In water; acetonitrile at 20℃; for 0.5h; Stage #3: With ammonium acetate In water 14; 15; 16; 24 FIG. 13 shows the oxidation of all-trans-RAL and all-trans-DRAL to all-trans-RA and all-trans-DRA, respectively; FIG. 24 shows the metabolism of all-trans-ROL and all-trans-DROL; RALDH Oxidation Assay. N-Acetylglucosaminyltransferase I-negative HEK-293S cells, obtained from Dr. G. Khorana (Massachusetts Institute of Technology, Boston) were cultured in Dulbecco's modified Eagle's medium, 10% fetal calf serum and maintained at 37° C., 5% CO2, and 100% humidity. Reeves, et al. Proc. Natl. Acad. Sci. U.S.A. 99:13419-13424, 2002. For RALDH enzyme assays, cells were transiently transfected with RALDH1, -2, -3, or -4 expression constructs using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. After 48 h post-transfection, the cells were collected by scraping and were centrifuged. The cell pellet was washed in 137 mM NaCl, 2.7 mM KCl, and 10 mM phosphate (pH 7.4), resuspended in 50 mM Tris (pH 8.0) containing 250 mM sucrose, and homogenized with the aid of a Dounce homogenizer. Cofactors were added to a final concentration of 5 mM NAD, 5 mM NADP, and 1 mM ATP. An aliquot of the cell lysate was boiled for 10 min at 95° C. to provide the control for the nonenzymatic reaction. Substrates in the form of all-trans-RAL or a mixture of isomers of DRAL were added to the cell lysates at a final concentration of 60 μM. The reactions were allowed to proceed for 2 h at 37° C. with shaking and were stopped by the addition of 2 volumes of CH3CN. Samples were treated for 30 min at room temperature with 100 mM NH2OH (final concentration from a freshly made stock of 1 M (pH 7.0)) followed by centrifugation at 12,000×g for 10 min. The clear supernatant was acidified with 0.1 volume of 0.5 M ammonium acetate (pH 4.0) and examined by reverse phase HPLC system (Zorbax ODS, 5 μm, 4.6×250 nm; Agilent, Foster City, Calif.) with an isocratic mobile phase A of 80% CH3CN, 10 mM ammonium acetate, 1% acetic acid, and a flow rate of 1.6 ml/min held for 15 min. After each run, the column was washed with mixture B (60% tert-butylmethyl ether, 40% methanol) for 10 min at 1.6 ml/min, followed by re-equilibration in phase A. The elution of RA and DRA isomers was monitored at 340 and 290 nm, respectively. The peaks were identified based on their spectra and coelution with standards. The cell lysate was examined for expression of RALDH1-4 by SDS-PAGE and immunoblotting of the V5 epitope-tagged recombinant protein using an anti-V5 epitope monoclonal antibody (Invitrogen); FIG. 24 shows the metabolism of all-trans-ROL and all-trans-DROL. RetSat saturates all-trans-ROL to all-trans-DROL, which was previously shown to be esterified by LRAT. Here we present evidence demonstrating that the oxidative metabolism of DROL closely follows that of ROL. Broad spectrum enzymes such as SDR and ADH carry out the reversible oxidation of all-trans-DROL to all-trans-DRAL. RALDH1, -2, -3, and -4 oxidize all-trans-DRAL to all-trans-DRA. Several members of the cytochrome P450 enzymes CYP26A1, -B1, and -C1 oxidize all-trans-DRA to all-trans-4-oxo-DRA, identified in vivo and in vitro. Other oxidized all-trans-DRA metabolites, which are not depicted, could be all-trans-4-hydroxy-DRA, all-trans-5,6-epoxy-DRA, all-trans-5,8-epoxy-DRA, and all-trans-18-hydroxy-DRA. The short-chain metabolite C19-ROL is shown here with its possible chemical structure. Its synthetic pathway may proceed from either all-trans-RA by decarboxylation and/or from all-trans-DRA via α-oxidation; Conversion of all-trans-DRAL to DRA is mediated by RALDH enzymes. Mouse RALDH1-4 cDNAs were cloned and fused at their C terminus with a tag containing a V5 epitope and His6 stretch. Glycosylation-deficient HEK-293S cells were transiently transfected with the tagged constructs of RALDH1, -2, -3, or -4 under the control of the CMV promoter. These cells allow the reproducible, high level expression of recombinant proteins. Reeves, et al. Proc. Natl. Acad. Sci. U.S.A. 99:13419-13424, 2002. The cell homogenate of transfected cells was supplemented with NAD, NADP, and ATP cofactors and with all-trans-RAL or all-trans-DRAL substrates. RALDH2 and -3 both efficiently converted all-trans-RAL and all-trans-DRAL into all-trans-RA and all-trans-DRA, respectively (FIGS. 13, A and B). The products all-trans-RA and all-trans-DRA were identified based on their elution time, absorbance spectra, and comparison with authentic standards (FIG. 13A, peak 1, and 13B, peak 6, and inset spectra). Other cis-DRA isomers were also produced as a result of oxidation of cis-DRAL isomers present in the synthetic mixture. The expression level of recombinant protein in transfected cell homogenate was verified by immunoblotting using anti-V5 monoclonal antibody for the presence of V5-tagged RALDH protein. This is shown for RALDH2-V5-His6 in FIG. 13 (top right panel). Based on the intensity of the immunoreactive band, similar expression levels of RALDH1, -2, -3, or -4 were attained in transfected cells. Homogenates of RALDH1- and RALDH4-transfected cells were less efficient in oxidizing all-trans-RAL or all-trans-DRAL, possibly a consequence of the C-terminal tag affecting some isozymes more than others. Alternatively, some isozymes maybe more active than others, as seen for mouse RALDH2 (Km=0.66 μm for all-trans-RAL) versus mouse RALDH1 (Km=11.6 μM for all-trans-RAL) (31, 32). Untransfected cells also exhibited significant activity toward both all-trans-RAL and all-trans-DRAL (FIG. 13, gray line chromatogram), suggesting endogenous RALDH activity in HEK-293S cells.FIG. 13 shows the oxidation of all-trans-RAL and all-trans-DRAL to all-trans-RA and all-trans-DRA, respectively. Cells were transiently transfected with vector carrying the cDNA of RALDH2 fused at its C terminus to a V5-His6 tag. The expression of RALDH2-V5-His6-tagged protein was confirmed by immunoblotting with anti-V5 monoclonal antibody and is shown in the top panel on the right in the lane labeled Raldh2. Cell homogenates of transfected HEK-RALDH2 cells (black solid line graph) or untransfected control cells (gray solid line graph) were incubated with all-trans-RAL (A) or all-trans-DRAL (B). Boiled control cells (black dashed line graph) were incubated with substrates under the same conditions. Retinoids were extracted and analyzed by reverse phase HPLC as described under “Methods and Materials.” The products of the reaction were identified based on their absorbance spectra and coelution with available standards. These are as follows: peak 1, all-trans-RA; peaks 2 and 3, syn- and anti-RAL oxime, respectively; peaks 4 and 5, cis-isomers of DRA; peak 6, all-trans-DRA; peaks 7-10, syn- and anti-oximes of several isomers of DRAL; peak 11, all-trans-DROL. The UV-visible absorbance spectra of peak 1 (identified as RA) and peak 6 (identified as DRA) are shown in middle and bottom panels on the right, respectively. The experiment was performed in duplicate and repeated three times. Similar results were obtained with cells transfected with RALDH3 tagged at the C terminus with V5-His6 tag.
Stage #1: all-trans-Retinal With NAD; NADP; ATP; 2-amino-2-hydroxymethyl-1,3-propanediol; Sucrose at 37℃; for 2h; Enzymatic reaction; Stage #2: With hydroxylamine In water; acetonitrile at 20℃; for 0.5h; Stage #3: With ammonium acetate In water 14; 15; 16; 24 FIG. 13 shows the oxidation of all-trans-RAL and all-trans-DRAL to all-trans-RA and all-trans-DRA, respectively; FIG. 24 shows the metabolism of all-trans-ROL and all-trans-DROL; RALDH Oxidation Assay. N-Acetylglucosaminyltransferase I-negative HEK-293S cells, obtained from Dr. G. Khorana (Massachusetts Institute of Technology, Boston) were cultured in Dulbecco's modified Eagle's medium, 10% fetal calf serum and maintained at 37° C., 5% CO2, and 100% humidity. Reeves, et al. Proc. Natl. Acad. Sci. U.S.A. 99:13419-13424, 2002. For RALDH enzyme assays, cells were transiently transfected with RALDH1, -2, -3, or -4 expression constructs using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. After 48 h post-transfection, the cells were collected by scraping and were centrifuged. The cell pellet was washed in 137 mM NaCl, 2.7 mM KCl, and 10 mM phosphate (pH 7.4), resuspended in 50 mM Tris (pH 8.0) containing 250 mM sucrose, and homogenized with the aid of a Dounce homogenizer. Cofactors were added to a final concentration of 5 mM NAD, 5 mM NADP, and 1 mM ATP. An aliquot of the cell lysate was boiled for 10 min at 95° C. to provide the control for the nonenzymatic reaction. Substrates in the form of all-trans-RAL or a mixture of isomers of DRAL were added to the cell lysates at a final concentration of 60 μM. The reactions were allowed to proceed for 2 h at 37° C. with shaking and were stopped by the addition of 2 volumes of CH3CN. Samples were treated for 30 min at room temperature with 100 mM NH2OH (final concentration from a freshly made stock of 1 M (pH 7.0)) followed by centrifugation at 12,000×g for 10 min. The clear supernatant was acidified with 0.1 volume of 0.5 M ammonium acetate (pH 4.0) and examined by reverse phase HPLC system (Zorbax ODS, 5 μm, 4.6×250 nm; Agilent, Foster City, Calif.) with an isocratic mobile phase A of 80% CH3CN, 10 mM ammonium acetate, 1% acetic acid, and a flow rate of 1.6 ml/min held for 15 min. After each run, the column was washed with mixture B (60% tert-butylmethyl ether, 40% methanol) for 10 min at 1.6 ml/min, followed by re-equilibration in phase A. The elution of RA and DRA isomers was monitored at 340 and 290 nm, respectively. The peaks were identified based on their spectra and coelution with standards. The cell lysate was examined for expression of RALDH1-4 by SDS-PAGE and immunoblotting of the V5 epitope-tagged recombinant protein using an anti-V5 epitope monoclonal antibody (Invitrogen); FIG. 24 shows the metabolism of all-trans-ROL and all-trans-DROL. RetSat saturates all-trans-ROL to all-trans-DROL, which was previously shown to be esterified by LRAT. Here we present evidence demonstrating that the oxidative metabolism of DROL closely follows that of ROL. Broad spectrum enzymes such as SDR and ADH carry out the reversible oxidation of all-trans-DROL to all-trans-DRAL. RALDH1, -2, -3, and -4 oxidize all-trans-DRAL to all-trans-DRA. Several members of the cytochrome P450 enzymes CYP26A1, -B1, and -C1 oxidize all-trans-DRA to all-trans-4-oxo-DRA, identified in vivo and in vitro. Other oxidized all-trans-DRA metabolites, which are not depicted, could be all-trans-4-hydroxy-DRA, all-trans-5,6-epoxy-DRA, all-trans-5,8-epoxy-DRA, and all-trans-18-hydroxy-DRA. The short-chain metabolite C19-ROL is shown here with its possible chemical structure. Its synthetic pathway may proceed from either all-trans-RA by decarboxylation and/or from all-trans-DRA via α-oxidation; Conversion of all-trans-DRAL to DRA is mediated by RALDH enzymes. Mouse RALDH1-4 cDNAs were cloned and fused at their C terminus with a tag containing a V5 epitope and His6 stretch. Glycosylation-deficient HEK-293S cells were transiently transfected with the tagged constructs of RALDH1, -2, -3, or -4 under the control of the CMV promoter. These cells allow the reproducible, high level expression of recombinant proteins. Reeves, et al. Proc. Natl. Acad. Sci. U.S.A. 99:13419-13424, 2002. The cell homogenate of transfected cells was supplemented with NAD, NADP, and ATP cofactors and with all-trans-RAL or all-trans-DRAL substrates. RALDH2 and -3 both efficiently converted all-trans-RAL and all-trans-DRAL into all-trans-RA and all-trans-DRA, respectively (FIGS. 13, A and B). The products all-trans-RA and all-trans-DRA were identified based on their elution time, absorbance spectra, and comparison with authentic standards (FIG. 13A, peak 1, and 13B, peak 6, and inset spectra). Other cis-DRA isomers were also produced as a result of oxidation of cis-DRAL isomers present in the synthetic mixture. The expression level of recombinant protein in transfected cell homogenate was verified by immunoblotting using anti-V5 monoclonal antibody for the presence of V5-tagged RALDH protein. This is shown for RALDH2-V5-His6 in FIG. 13 (top right panel). Based on the intensity of the immunoreactive band, similar expression levels of RALDH1, -2, -3, or -4 were attained in transfected cells. Homogenates of RALDH1- and RALDH4-transfected cells were less efficient in oxidizing all-trans-RAL or all-trans-DRAL, possibly a consequence of the C-terminal tag affecting some isozymes more than others. Alternatively, some isozymes maybe more active than others, as seen for mouse RALDH2 (Km=0.66 μm for all-trans-RAL) versus mouse RALDH1 (Km=11.6 μM for all-trans-RAL) (31, 32). Untransfected cells also exhibited significant activity toward both all-trans-RAL and all-trans-DRAL (FIG. 13, gray line chromatogram), suggesting endogenous RALDH activity in HEK-293S cells.FIG. 13 shows the oxidation of all-trans-RAL and all-trans-DRAL to all-trans-RA and all-trans-DRA, respectively. Cells were transiently transfected with vector carrying the cDNA of RALDH2 fused at its C terminus to a V5-His6 tag. The expression of RALDH2-V5-His6-tagged protein was confirmed by immunoblotting with anti-V5 monoclonal antibody and is shown in the top panel on the right in the lane labeled Raldh2. Cell homogenates of transfected HEK-RALDH2 cells (black solid line graph) or untransfected control cells (gray solid line graph) were incubated with all-trans-RAL (A) or all-trans-DRAL (B). Boiled control cells (black dashed line graph) were incubated with substrates under the same conditions. Retinoids were extracted and analyzed by reverse phase HPLC as described under “Methods and Materials.” The products of the reaction were identified based on their absorbance spectra and coelution with available standards. These are as follows: peak 1, all-trans-RA; peaks 2 and 3, syn- and anti-RAL oxime, respectively; peaks 4 and 5, cis-isomers of DRA; peak 6, all-trans-DRA; peaks 7-10, syn- and anti-oximes of several isomers of DRAL; peak 11, all-trans-DROL. The UV-visible absorbance spectra of peak 1 (identified as RA) and peak 6 (identified as DRA) are shown in middle and bottom panels on the right, respectively. The experiment was performed in duplicate and repeated three times. Similar results were obtained with cells transfected with RALDH3 tagged at the C terminus with V5-His6 tag.
Stage #1: all-trans-Retinal With NAD; NADP; ATP; 2-amino-2-hydroxymethyl-1,3-propanediol; Sucrose at 37℃; for 2h; Enzymatic reaction; Stage #2: With hydroxylamine In water; acetonitrile at 20℃; for 0.5h; Stage #3: With ammonium acetate In water 14; 15; 16; 24 FIG. 13 shows the oxidation of all-trans-RAL and all-trans-DRAL to all-trans-RA and all-trans-DRA, respectively; FIG. 24 shows the metabolism of all-trans-ROL and all-trans-DROL; RALDH Oxidation Assay. N-Acetylglucosaminyltransferase I-negative HEK-293S cells, obtained from Dr. G. Khorana (Massachusetts Institute of Technology, Boston) were cultured in Dulbecco's modified Eagle's medium, 10% fetal calf serum and maintained at 37° C., 5% CO2, and 100% humidity. Reeves, et al. Proc. Natl. Acad. Sci. U.S.A. 99:13419-13424, 2002. For RALDH enzyme assays, cells were transiently transfected with RALDH1, -2, -3, or -4 expression constructs using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. After 48 h post-transfection, the cells were collected by scraping and were centrifuged. The cell pellet was washed in 137 mM NaCl, 2.7 mM KCl, and 10 mM phosphate (pH 7.4), resuspended in 50 mM Tris (pH 8.0) containing 250 mM sucrose, and homogenized with the aid of a Dounce homogenizer. Cofactors were added to a final concentration of 5 mM NAD, 5 mM NADP, and 1 mM ATP. An aliquot of the cell lysate was boiled for 10 min at 95° C. to provide the control for the nonenzymatic reaction. Substrates in the form of all-trans-RAL or a mixture of isomers of DRAL were added to the cell lysates at a final concentration of 60 μM. The reactions were allowed to proceed for 2 h at 37° C. with shaking and were stopped by the addition of 2 volumes of CH3CN. Samples were treated for 30 min at room temperature with 100 mM NH2OH (final concentration from a freshly made stock of 1 M (pH 7.0)) followed by centrifugation at 12,000×g for 10 min. The clear supernatant was acidified with 0.1 volume of 0.5 M ammonium acetate (pH 4.0) and examined by reverse phase HPLC system (Zorbax ODS, 5 μm, 4.6×250 nm; Agilent, Foster City, Calif.) with an isocratic mobile phase A of 80% CH3CN, 10 mM ammonium acetate, 1% acetic acid, and a flow rate of 1.6 ml/min held for 15 min. After each run, the column was washed with mixture B (60% tert-butylmethyl ether, 40% methanol) for 10 min at 1.6 ml/min, followed by re-equilibration in phase A. The elution of RA and DRA isomers was monitored at 340 and 290 nm, respectively. The peaks were identified based on their spectra and coelution with standards. The cell lysate was examined for expression of RALDH1-4 by SDS-PAGE and immunoblotting of the V5 epitope-tagged recombinant protein using an anti-V5 epitope monoclonal antibody (Invitrogen); FIG. 24 shows the metabolism of all-trans-ROL and all-trans-DROL. RetSat saturates all-trans-ROL to all-trans-DROL, which was previously shown to be esterified by LRAT. Here we present evidence demonstrating that the oxidative metabolism of DROL closely follows that of ROL. Broad spectrum enzymes such as SDR and ADH carry out the reversible oxidation of all-trans-DROL to all-trans-DRAL. RALDH1, -2, -3, and -4 oxidize all-trans-DRAL to all-trans-DRA. Several members of the cytochrome P450 enzymes CYP26A1, -B1, and -C1 oxidize all-trans-DRA to all-trans-4-oxo-DRA, identified in vivo and in vitro. Other oxidized all-trans-DRA metabolites, which are not depicted, could be all-trans-4-hydroxy-DRA, all-trans-5,6-epoxy-DRA, all-trans-5,8-epoxy-DRA, and all-trans-18-hydroxy-DRA. The short-chain metabolite C19-ROL is shown here with its possible chemical structure. Its synthetic pathway may proceed from either all-trans-RA by decarboxylation and/or from all-trans-DRA via α-oxidation; Conversion of all-trans-DRAL to DRA is mediated by RALDH enzymes. Mouse RALDH1-4 cDNAs were cloned and fused at their C terminus with a tag containing a V5 epitope and His6 stretch. Glycosylation-deficient HEK-293S cells were transiently transfected with the tagged constructs of RALDH1, -2, -3, or -4 under the control of the CMV promoter. These cells allow the reproducible, high level expression of recombinant proteins. Reeves, et al. Proc. Natl. Acad. Sci. U.S.A. 99:13419-13424, 2002. The cell homogenate of transfected cells was supplemented with NAD, NADP, and ATP cofactors and with all-trans-RAL or all-trans-DRAL substrates. RALDH2 and -3 both efficiently converted all-trans-RAL and all-trans-DRAL into all-trans-RA and all-trans-DRA, respectively (FIGS. 13, A and B). The products all-trans-RA and all-trans-DRA were identified based on their elution time, absorbance spectra, and comparison with authentic standards (FIG. 13A, peak 1, and 13B, peak 6, and inset spectra). Other cis-DRA isomers were also produced as a result of oxidation of cis-DRAL isomers present in the synthetic mixture. The expression level of recombinant protein in transfected cell homogenate was verified by immunoblotting using anti-V5 monoclonal antibody for the presence of V5-tagged RALDH protein. This is shown for RALDH2-V5-His6 in FIG. 13 (top right panel). Based on the intensity of the immunoreactive band, similar expression levels of RALDH1, -2, -3, or -4 were attained in transfected cells. Homogenates of RALDH1- and RALDH4-transfected cells were less efficient in oxidizing all-trans-RAL or all-trans-DRAL, possibly a consequence of the C-terminal tag affecting some isozymes more than others. Alternatively, some isozymes maybe more active than others, as seen for mouse RALDH2 (Km=0.66 μm for all-trans-RAL) versus mouse RALDH1 (Km=11.6 μM for all-trans-RAL) (31, 32). Untransfected cells also exhibited significant activity toward both all-trans-RAL and all-trans-DRAL (FIG. 13, gray line chromatogram), suggesting endogenous RALDH activity in HEK-293S cells.FIG. 13 shows the oxidation of all-trans-RAL and all-trans-DRAL to all-trans-RA and all-trans-DRA, respectively. Cells were transiently transfected with vector carrying the cDNA of RALDH2 fused at its C terminus to a V5-His6 tag. The expression of RALDH2-V5-His6-tagged protein was confirmed by immunoblotting with anti-V5 monoclonal antibody and is shown in the top panel on the right in the lane labeled Raldh2. Cell homogenates of transfected HEK-RALDH2 cells (black solid line graph) or untransfected control cells (gray solid line graph) were incubated with all-trans-RAL (A) or all-trans-DRAL (B). Boiled control cells (black dashed line graph) were incubated with substrates under the same conditions. Retinoids were extracted and analyzed by reverse phase HPLC as described under “Methods and Materials.” The products of the reaction were identified based on their absorbance spectra and coelution with available standards. These are as follows: peak 1, all-trans-RA; peaks 2 and 3, syn- and anti-RAL oxime, respectively; peaks 4 and 5, cis-isomers of DRA; peak 6, all-trans-DRA; peaks 7-10, syn- and anti-oximes of several isomers of DRAL; peak 11, all-trans-DROL. The UV-visible absorbance spectra of peak 1 (identified as RA) and peak 6 (identified as DRA) are shown in middle and bottom panels on the right, respectively. The experiment was performed in duplicate and repeated three times. Similar results were obtained with cells transfected with RALDH3 tagged at the C terminus with V5-His6 tag.
Stage #1: all-trans-Retinal With NAD; NADP; ATP; 2-amino-2-hydroxymethyl-1,3-propanediol; Sucrose at 37℃; for 2h; Enzymatic reaction; Stage #2: With hydroxylamine In water; acetonitrile at 20℃; for 0.5h; Stage #3: With ammonium acetate In water 14; 15; 16; 24 FIG. 13 shows the oxidation of all-trans-RAL and all-trans-DRAL to all-trans-RA and all-trans-DRA, respectively; FIG. 24 shows the metabolism of all-trans-ROL and all-trans-DROL; RALDH Oxidation Assay. N-Acetylglucosaminyltransferase I-negative HEK-293S cells, obtained from Dr. G. Khorana (Massachusetts Institute of Technology, Boston) were cultured in Dulbecco's modified Eagle's medium, 10% fetal calf serum and maintained at 37° C., 5% CO2, and 100% humidity. Reeves, et al. Proc. Natl. Acad. Sci. U.S.A. 99:13419-13424, 2002. For RALDH enzyme assays, cells were transiently transfected with RALDH1, -2, -3, or -4 expression constructs using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. After 48 h post-transfection, the cells were collected by scraping and were centrifuged. The cell pellet was washed in 137 mM NaCl, 2.7 mM KCl, and 10 mM phosphate (pH 7.4), resuspended in 50 mM Tris (pH 8.0) containing 250 mM sucrose, and homogenized with the aid of a Dounce homogenizer. Cofactors were added to a final concentration of 5 mM NAD, 5 mM NADP, and 1 mM ATP. An aliquot of the cell lysate was boiled for 10 min at 95° C. to provide the control for the nonenzymatic reaction. Substrates in the form of all-trans-RAL or a mixture of isomers of DRAL were added to the cell lysates at a final concentration of 60 μM. The reactions were allowed to proceed for 2 h at 37° C. with shaking and were stopped by the addition of 2 volumes of CH3CN. Samples were treated for 30 min at room temperature with 100 mM NH2OH (final concentration from a freshly made stock of 1 M (pH 7.0)) followed by centrifugation at 12,000×g for 10 min. The clear supernatant was acidified with 0.1 volume of 0.5 M ammonium acetate (pH 4.0) and examined by reverse phase HPLC system (Zorbax ODS, 5 μm, 4.6×250 nm; Agilent, Foster City, Calif.) with an isocratic mobile phase A of 80% CH3CN, 10 mM ammonium acetate, 1% acetic acid, and a flow rate of 1.6 ml/min held for 15 min. After each run, the column was washed with mixture B (60% tert-butylmethyl ether, 40% methanol) for 10 min at 1.6 ml/min, followed by re-equilibration in phase A. The elution of RA and DRA isomers was monitored at 340 and 290 nm, respectively. The peaks were identified based on their spectra and coelution with standards. The cell lysate was examined for expression of RALDH1-4 by SDS-PAGE and immunoblotting of the V5 epitope-tagged recombinant protein using an anti-V5 epitope monoclonal antibody (Invitrogen); FIG. 24 shows the metabolism of all-trans-ROL and all-trans-DROL. RetSat saturates all-trans-ROL to all-trans-DROL, which was previously shown to be esterified by LRAT. Here we present evidence demonstrating that the oxidative metabolism of DROL closely follows that of ROL. Broad spectrum enzymes such as SDR and ADH carry out the reversible oxidation of all-trans-DROL to all-trans-DRAL. RALDH1, -2, -3, and -4 oxidize all-trans-DRAL to all-trans-DRA. Several members of the cytochrome P450 enzymes CYP26A1, -B1, and -C1 oxidize all-trans-DRA to all-trans-4-oxo-DRA, identified in vivo and in vitro. Other oxidized all-trans-DRA metabolites, which are not depicted, could be all-trans-4-hydroxy-DRA, all-trans-5,6-epoxy-DRA, all-trans-5,8-epoxy-DRA, and all-trans-18-hydroxy-DRA. The short-chain metabolite C19-ROL is shown here with its possible chemical structure. Its synthetic pathway may proceed from either all-trans-RA by decarboxylation and/or from all-trans-DRA via α-oxidation; Conversion of all-trans-DRAL to DRA is mediated by RALDH enzymes. Mouse RALDH1-4 cDNAs were cloned and fused at their C terminus with a tag containing a V5 epitope and His6 stretch. Glycosylation-deficient HEK-293S cells were transiently transfected with the tagged constructs of RALDH1, -2, -3, or -4 under the control of the CMV promoter. These cells allow the reproducible, high level expression of recombinant proteins. Reeves, et al. Proc. Natl. Acad. Sci. U.S.A. 99:13419-13424, 2002. The cell homogenate of transfected cells was supplemented with NAD, NADP, and ATP cofactors and with all-trans-RAL or all-trans-DRAL substrates. RALDH2 and -3 both efficiently converted all-trans-RAL and all-trans-DRAL into all-trans-RA and all-trans-DRA, respectively (FIGS. 13, A and B). The products all-trans-RA and all-trans-DRA were identified based on their elution time, absorbance spectra, and comparison with authentic standards (FIG. 13A, peak 1, and 13B, peak 6, and inset spectra). Other cis-DRA isomers were also produced as a result of oxidation of cis-DRAL isomers present in the synthetic mixture. The expression level of recombinant protein in transfected cell homogenate was verified by immunoblotting using anti-V5 monoclonal antibody for the presence of V5-tagged RALDH protein. This is shown for RALDH2-V5-His6 in FIG. 13 (top right panel). Based on the intensity of the immunoreactive band, similar expression levels of RALDH1, -2, -3, or -4 were attained in transfected cells. Homogenates of RALDH1- and RALDH4-transfected cells were less efficient in oxidizing all-trans-RAL or all-trans-DRAL, possibly a consequence of the C-terminal tag affecting some isozymes more than others. Alternatively, some isozymes maybe more active than others, as seen for mouse RALDH2 (Km=0.66 μm for all-trans-RAL) versus mouse RALDH1 (Km=11.6 μM for all-trans-RAL) (31, 32). Untransfected cells also exhibited significant activity toward both all-trans-RAL and all-trans-DRAL (FIG. 13, gray line chromatogram), suggesting endogenous RALDH activity in HEK-293S cells.FIG. 13 shows the oxidation of all-trans-RAL and all-trans-DRAL to all-trans-RA and all-trans-DRA, respectively. Cells were transiently transfected with vector carrying the cDNA of RALDH2 fused at its C terminus to a V5-His6 tag. The expression of RALDH2-V5-His6-tagged protein was confirmed by immunoblotting with anti-V5 monoclonal antibody and is shown in the top panel on the right in the lane labeled Raldh2. Cell homogenates of transfected HEK-RALDH2 cells (black solid line graph) or untransfected control cells (gray solid line graph) were incubated with all-trans-RAL (A) or all-trans-DRAL (B). Boiled control cells (black dashed line graph) were incubated with substrates under the same conditions. Retinoids were extracted and analyzed by reverse phase HPLC as described under “Methods and Materials.” The products of the reaction were identified based on their absorbance spectra and coelution with available standards. These are as follows: peak 1, all-trans-RA; peaks 2 and 3, syn- and anti-RAL oxime, respectively; peaks 4 and 5, cis-isomers of DRA; peak 6, all-trans-DRA; peaks 7-10, syn- and anti-oximes of several isomers of DRAL; peak 11, all-trans-DROL. The UV-visible absorbance spectra of peak 1 (identified as RA) and peak 6 (identified as DRA) are shown in middle and bottom panels on the right, respectively. The experiment was performed in duplicate and repeated three times. Similar results were obtained with cells transfected with RALDH3 tagged at the C terminus with V5-His6 tag.
Stage #1: all-trans-Retinal With NAD; NADP; ATP; 2-amino-2-hydroxymethyl-1,3-propanediol; Sucrose at 37℃; for 2h; Enzymatic reaction; Stage #2: With hydroxylamine In water; acetonitrile at 20℃; for 0.5h; Stage #3: With ammonium acetate In water 14; 16 FIG. 13 shows the oxidation of all-trans-RAL and all-trans-DRAL to all-trans-RA and all-trans-DRA, respectively; RALDH Oxidation Assay. N-Acetylglucosaminyltransferase I-negative HEK-293S cells, obtained from Dr. G. Khorana (Massachusetts Institute of Technology, Boston) were cultured in Dulbecco's modified Eagle's medium, 10% fetal calf serum and maintained at 37° C., 5% CO2, and 100% humidity. Reeves, et al. Proc. Natl. Acad. Sci. U.S.A. 99:13419-13424, 2002. For RALDH enzyme assays, cells were transiently transfected with RALDH1, -2, -3, or -4 expression constructs using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. After 48 h post-transfection, the cells were collected by scraping and were centrifuged. The cell pellet was washed in 137 mM NaCl, 2.7 mM KCl, and 10 mM phosphate (pH 7.4), resuspended in 50 mM Tris (pH 8.0) containing 250 mM sucrose, and homogenized with the aid of a Dounce homogenizer. Cofactors were added to a final concentration of 5 mM NAD, 5 mM NADP, and 1 mM ATP. An aliquot of the cell lysate was boiled for 10 min at 95° C. to provide the control for the nonenzymatic reaction. Substrates in the form of all-trans-RAL or a mixture of isomers of DRAL were added to the cell lysates at a final concentration of 60 μM. The reactions were allowed to proceed for 2 h at 37° C. with shaking and were stopped by the addition of 2 volumes of CH3CN. Samples were treated for 30 min at room temperature with 100 mM NH2OH (final concentration from a freshly made stock of 1 M (pH 7.0)) followed by centrifugation at 12,000×g for 10 min. The clear supernatant was acidified with 0.1 volume of 0.5 M ammonium acetate (pH 4.0) and examined by reverse phase HPLC system (Zorbax ODS, 5 μm, 4.6×250 nm; Agilent, Foster City, Calif.) with an isocratic mobile phase A of 80% CH3CN, 10 mM ammonium acetate, 1% acetic acid, and a flow rate of 1.6 ml/min held for 15 min. After each run, the column was washed with mixture B (60% tert-butylmethyl ether, 40% methanol) for 10 min at 1.6 ml/min, followed by re-equilibration in phase A. The elution of RA and DRA isomers was monitored at 340 and 290 nm, respectively. The peaks were identified based on their spectra and coelution with standards. The cell lysate was examined for expression of RALDH1-4 by SDS-PAGE and immunoblotting of the V5 epitope-tagged recombinant protein using an anti-V5 epitope monoclonal antibody (Invitrogen); Conversion of all-trans-DRAL to DRA is mediated by RALDH enzymes. Mouse RALDH1-4 cDNAs were cloned and fused at their C terminus with a tag containing a V5 epitope and His6 stretch. Glycosylation-deficient HEK-293S cells were transiently transfected with the tagged constructs of RALDH1, -2, -3, or -4 under the control of the CMV promoter. These cells allow the reproducible, high level expression of recombinant proteins. Reeves, et al. Proc. Natl. Acad. Sci. U.S.A. 99:13419-13424, 2002. The cell homogenate of transfected cells was supplemented with NAD, NADP, and ATP cofactors and with all-trans-RAL or all-trans-DRAL substrates. RALDH2 and -3 both efficiently converted all-trans-RAL and all-trans-DRAL into all-trans-RA and all-trans-DRA, respectively (FIGS. 13, A and B). The products all-trans-RA and all-trans-DRA were identified based on their elution time, absorbance spectra, and comparison with authentic standards (FIG. 13A, peak 1, and 13B, peak 6, and inset spectra). Other cis-DRA isomers were also produced as a result of oxidation of cis-DRAL isomers present in the synthetic mixture. The expression level of recombinant protein in transfected cell homogenate was verified by immunoblotting using anti-V5 monoclonal antibody for the presence of V5-tagged RALDH protein. This is shown for RALDH2-V5-His6 in FIG. 13 (top right panel). Based on the intensity of the immunoreactive band, similar expression levels of RALDH1, -2, -3, or -4 were attained in transfected cells. Homogenates of RALDH1- and RALDH4-transfected cells were less efficient in oxidizing all-trans-RAL or all-trans-DRAL, possibly a consequence of the C-terminal tag affecting some isozymes more than others. Alternatively, some isozymes maybe more active than others, as seen for mouse RALDH2 (Km=0.66 μm for all-trans-RAL) versus mouse RALDH1 (Km=11.6 μM for all-trans-RAL) (31, 32). Untransfected cells also exhibited significant activity toward both all-trans-RAL and all-trans-DRAL (FIG. 13, gray line chromatogram), suggesting endogenous RALDH activity in HEK-293S cells.FIG. 13 shows the oxidation of all-trans-RAL and all-trans-DRAL to all-trans-RA and all-trans-DRA, respectively. Cells were transiently transfected with vector carrying the cDNA of RALDH2 fused at its C terminus to a V5-His6 tag. The expression of RALDH2-V5-His6-tagged protein was confirmed by immunoblotting with anti-V5 monoclonal antibody and is shown in the top panel on the right in the lane labeled Raldh2. Cell homogenates of transfected HEK-RALDH2 cells (black solid line graph) or untransfected control cells (gray solid line graph) were incubated with all-trans-RAL (A) or all-trans-DRAL (B). Boiled control cells (black dashed line graph) were incubated with substrates under the same conditions. Retinoids were extracted and analyzed by reverse phase HPLC as described under “Methods and Materials.” The products of the reaction were identified based on their absorbance spectra and coelution with available standards. These are as follows: peak 1, all-trans-RA; peaks 2 and 3, syn- and anti-RAL oxime, respectively; peaks 4 and 5, cis-isomers of DRA; peak 6, all-trans-DRA; peaks 7-10, syn- and anti-oximes of several isomers of DRAL; peak 11, all-trans-DROL. The UV-visible absorbance spectra of peak 1 (identified as RA) and peak 6 (identified as DRA) are shown in middle and bottom panels on the right, respectively. The experiment was performed in duplicate and repeated three times. Similar results were obtained with cells transfected with RALDH3 tagged at the C terminus with V5-His6 tag.

  • 70
  • [ 116-31-4 ]
  • [ 4759-48-2 ]
  • [ 302-79-4 ]
  • [ 302-79-4 ]
YieldReaction ConditionsOperation in experiment
tetracycline-induced HEKK-mouse retinol saturase (RetSat) cells;Enzymatic reaction;Product distribution / selectivity; FIG. 7 shows RetSat activity towards all-trans-retinoic acid; Incubation of cells with retinoic acid indicated that it is not substrate for saturation by RetSat (FIG. 7A). Synthetic 13-14-dihydroretinoic acid standards were examined on the same HPLC system to establish their elution conditions (FIG. 7B). Even though, 13-cis retinoic acid (peak 1, FIG. 7A) coelutes with all-trans-13,14-dihydroretinoic (peak 7, FIG. 7B) the absorbance spectrum of the two compounds is different (FIGS. 7A and B insets) and allowed us to conclude that 13,14-dihydroretinoic acid cannot be detected in RetSat-expressing cells incubated with retinoic acid.FIG. 7 shows RetSat activity towards all-trans-retinoic acid. (A) Analysis of retinoic acid conversion in RetSat-expressing cells. Tet-induced HEKK-RetSat or untransfected cells were incubated overnight with pure all-trans-retinoic acid (>90% pure by HPLC, assayed before incubation). Following incubation retinoic acid was extracted and analyzed by reverse-phase HPLC System II. The appearance of 13,14-dihydroretinoic acid isomers was monitored at 290 nm (expected 25-30 minutes after injection). Peak numbers represent 13-cis-retinoic acid, 9,13-di-cis-retinoic acid, 9-cis-retinoic acid and all-trans-retinoic acid. Ruiz, et al. J Biol Chem 274:3834-3841, 1999; Batten, et al. J Biol Chem 279:10422-10432, 2004; Kuksa, et al. Vision Res 43:2959-2981, 2003; Imanishi, et al. J Cell Biol 164:373-383, 2004. (B) Mixture of isomers of synthetic standards of 13,14-dihydroretinoic acid were examined by reverse-phase HPLC System II in order to establish product elution profile. Inset shows the spectra of the different isomers of 13,14-dihydroretinoic acid. Star (*) indicates an unrelated compound. The experiment was performed in triplicate samples and repeated.
untransfected HEKK cells;Enzymatic reaction;Product distribution / selectivity; FIG. 7 shows RetSat activity towards all-trans-retinoic acid; Incubation of cells with retinoic acid indicated that it is not substrate for saturation by RetSat (FIG. 7A). Synthetic 13-14-dihydroretinoic acid standards were examined on the same HPLC system to establish their elution conditions (FIG. 7B). Even though, 13-cis retinoic acid (peak 1, FIG. 7A) coelutes with all-trans-13,14-dihydroretinoic (peak 7, FIG. 7B) the absorbance spectrum of the two compounds is different (FIGS. 7A and B insets) and allowed us to conclude that 13,14-dihydroretinoic acid cannot be detected in RetSat-expressing cells incubated with retinoic acid.FIG. 7 shows RetSat activity towards all-trans-retinoic acid. (A) Analysis of retinoic acid conversion in RetSat-expressing cells. Tet-induced HEKK-RetSat or untransfected cells were incubated overnight with pure all-trans-retinoic acid (>90% pure by HPLC, assayed before incubation). Following incubation retinoic acid was extracted and analyzed by reverse-phase HPLC System II. The appearance of 13,14-dihydroretinoic acid isomers was monitored at 290 nm (expected 25-30 minutes after injection). Peak numbers represent 13-cis-retinoic acid, 9,13-di-cis-retinoic acid, 9-cis-retinoic acid and all-trans-retinoic acid. Ruiz, et al. J Biol Chem 274:3834-3841, 1999; Batten, et al. J Biol Chem 279:10422-10432, 2004; Kuksa, et al. Vision Res 43:2959-2981, 2003; Imanishi, et al. J Cell Biol 164:373-383, 2004. (B) Mixture of isomers of synthetic standards of 13,14-dihydroretinoic acid were examined by reverse-phase HPLC System II in order to establish product elution profile. Inset shows the spectra of the different isomers of 13,14-dihydroretinoic acid. Star (*) indicates an unrelated compound. The experiment was performed in triplicate samples and repeated.
  • 71
  • 13-acetyl-17,18-dihydro-10-mesityl-18,18-dimethylporphyrin [ No CAS ]
  • [ 116-31-4 ]
  • 13-[(2E,4E,6E,8E,10E)-5,9-dimethyl-11-(2,6,6-trimethylcyclohex-1-enyl)undeca-2,4,6,8,10-pentaen-1-onyl]-17,18-dihydro-10-mesityl-18,18-dimethylporphyrin [ No CAS ]
YieldReaction ConditionsOperation in experiment
53% With sodium hydroxide In ethanol at 20 - 80℃; for 0.666667h; Microwave irradiation;
  • 72
  • [ 1222538-51-3 ]
  • [ 472-86-6 ]
  • [ 116-31-4 ]
  • 73
  • [ 564-87-4 ]
  • [ 472-86-6 ]
  • [ 116-31-4 ]
  • 74
  • [ 116-31-4 ]
  • [ 141-43-5 ]
  • [ 173449-96-2 ]
YieldReaction ConditionsOperation in experiment
51% With acetic acid In dichloromethane at 20℃; for 72h; Darkness;
at 20℃; for 48h;
With acetic acid In ethanol at 20℃; for 72h; Darkness;
With acetic acid In ethanol at 20 - 25℃; Darkness; Inert atmosphere; A2E Synthesis A2E was synthesized as described by Parish et al. (Parish etal. 1998) with modification to the purification methods. Twoequivalents of all-trans-retinal and one equivalent of ethano65 lamine were mixed together in ethanol with one equivalent ofacetic acid. The mixture was placed in the dark at roomtemperature (20-25° C.) for 3-4 days. The solvent was evapoThe rated with argon gas, and A2E was separated from the reaction mixture by HPLC with separation column (Synergi 4 tm Hydro-RP 80A 250x 10.00mm). Isocratic gradient of 3% of H20 (0.1% formic acid) and 97% methanol was applied for the separation. The reaction mixture (100 pL) was injected for 60 minutes with a flow rate of 1.0 mE/mm. The retention time of A2E and iso-A2E was approximately 23 minutes. The absorption spectrum was monitored at 430 nm. A2E was verified with an ultraviolet-visible absorption spectrophotometer and mass spectrometry.
With acetic acid In ethanol at 20℃; for 72h; Darkness; 4.3. Synthesis of A2E A2E was synthesized from all-trans-retinal and ethanolamine [35]. Mixtures of all-trans-retinal, ethanolamine, and acetic acid in ethanol were stirred in the dark, at room temperature, for 72 h. The mixture was purified by silica gel column chromatography. The mixture was loaded in a silica gel column that was prewashed with a mixed solvent (methanol: dichloromethane = 5:95). Then, A2E was eluted with a mixed solvent (methanol: dichloromethane = 8:92) containing 0.02% TFA. After separation, A2E was concentrated under nitrogen gas in a concentrator and purified to a single peak using Ultimate 3000 HPLC system (Dionex Corporation, Sunnyvale, CA, USA) with an automated fraction collector system (AFC-3000 UltiMate Fraction Collector; Thermo-Fisher Scientific, Waltham, MA, USA). The purified A2E was confirmed by a chromatogram and absorbance spectrum using HPLC and spectrophotometer, respectively. A2E stock solution was 10 mM in DMSO and was stored at -20 °C in the dark. In each experiment, A2E stock solution was diluted in a culture media at concentration of 10 and 25 μM.

  • 75
  • [ 116-31-4 ]
  • [ 472-86-6 ]
  • [ 116-31-4 ]
  • 76
  • [ 68-26-8 ]
  • [ 472-86-6 ]
  • [ 116-31-4 ]
  • 77
  • [ 116-31-4 ]
  • [ 1779-49-3 ]
  • (1E,3E,5E,7E)-2-(3,7-dimethyldeca-1,3,5,7,9-pentaenyl)-1,3,3-trimethylcyclohex-1-ene [ No CAS ]
YieldReaction ConditionsOperation in experiment
85% Stage #1: Methyltriphenylphosphonium bromide With sodium hexamethyldisilazane In tetrahydrofuran; hexane at 25℃; for 0.5h; Inert atmosphere; Stage #2: all-trans-Retinal With N,N,N,N,N,N-hexamethylphosphoric triamide In tetrahydrofuran; hexane at -78℃; for 1.5h; Inert atmosphere;
  • 78
  • [ 127-47-9 ]
  • [ 472-86-6 ]
  • [ 116-31-4 ]
  • 79
  • [ 108-73-6 ]
  • [ 116-31-4 ]
  • [ 1641587-37-2 ]
YieldReaction ConditionsOperation in experiment
With acetic acid In ethanol at 20℃; for 48h; Darkness;
With acetic acid In ethanol at 20℃; for 48h; Darkness; 1 2-methyl-2-((1 E,3E,5£)-4-methyl-6-(2,6,6-trimethylcyclohex-1 -enyl)hexa- 1 ,3,5-trienyl)-2H-chromene-5,7-diol ; Chromene A To a stirred solution of trans- retinal (200 mg, 0.35 mmol) in ethanol (8 ml_), were added phloroglucinol (48.28 mg, 0.35 mmol) and acetic acid (40 μΙ_, 0.35 mmol). The reaction was stirred at room temperature for 48h, protected from the light with foil paper. After concentration of solvent under reduced pressure, the residue obtained was dissolved in AcOEt, (20 ml_) and washed with water (10 ml_). The organic layer was recovered, dried with MgS04 and concentrated under reduced pressure. The residue obtained was purified by chromatography on silica gel (90/10 to 85/15 pentane/ AcOEt) to give Chromene A (1 12.4 mg, 41 %) as a solid contaminated by 13% of a by product. Chromene A was isolated after purification by preparative HPLC to furnish full characterization (gradient of hexane/AcOEt, t0 15 mL/min, column luna 5μ Silica 100A 250x21 .20 mm, detection 254 nm). (0258) Ft, (CH2CI2/MeOH) 0.4; 1 H NMR (500 MHz; CD3OD) δΗ 6.63 (dd, J = 1 1 .5 Hz, J = 15.5 Hz, 1 H, Hi i ), 6.63 (d, J = 10.0 Hz, 1 H, H15), 6.14 (d, J = 16.5 Hz, 1 H, H7), 6.03 (d, J = 16.5 Hz, 1 H, H8), 5.98 (d, J= 1 1 .0 Hz, 1 H, H10), 5.85 and 5.81 (d, J = 2.0 Hz, 1 H, and d, J = 2.5 Hz, 1 H, H18 and H20), 5.75 (d, J = 15.0 Hz, 1 H, H12), 5.39 (d, J = 10.0 Hz, 1 H, H14), 2.02-2.00 (m, 2H, H4 (CH2)), 1 .86 (s, 3H, H25 (CH3)), 1 .68 (s, 3H, H24 (CH3)), 1 .67-1 .60 (m, 2H, H3 (CH2)), 1 .49 (s, 3H, H26 (CH3)), 1 .48-1 .46 (m, 2H, H2 (CH2)), 1 .00 (s, 6H, H22, H23 (CH3)); 13C NMR (125 MHz; CDCI3 5C 159.7 (C19), 156.4 (C21/17), 139.1 (C8), 139.0 (C6), 137.1 (C12), 137.0 (C9), 130.4 (C10), 129.9 (C5), 127.6 (C7), 126.3 (C15), 123.1 (C14), 1 19.1 (C,,), 103.9 (C16), 96.3 (C20/i8) , 78.5 (C13), 40.7 (C2), 35.1 (d ), 33.9 (C4), 29.3 (C22/23), 27.6 (C26), 21 .8 (C24), 20.3 (C3), 12.6 (C25); HRMS (ESI-TOF) m/z: [M-H]" calcd. for C26H3103 391 .2278; found 391 .2272; HPLC rt: 1 1 .26 min, (Atlantis C18 5μπι (4.6x250 mm), H20 0.1 %TFA ACN, (0259) , detection 298 nm).
  • 80
  • [ 116-31-4 ]
  • [ 141-43-5 ]
  • N-retinylidene-N-retinylethanolamine [ No CAS ]
YieldReaction ConditionsOperation in experiment
Stage #1: all-trans-Retinal; ethanolamine With acetic acid In ethanol at 20℃; for 48h; Stage #2: With trifluoroacetic acid In methanol; dichloromethane Stage #3: With sodium chloride In dichloromethane 1 Example 1: Synthesis of A2E A2E was synthesized by a method known from the existing literature (Proc. Natl. Acad Sci. USA Vol. 95, pp. 14609-14613). Specifically, all-trans-retinal (100 mg, 352 mmol) And ethanolamine (9.5 mg, 155 mmol) were added to ethanol (3.0 ml), and the mixture was stirred at room temperature for 2 days in the presence of acetic acid (9.3 ml, 155 mmol) and stirred. The reaction was concentrated under reduced pressure And separated by silica gel chromatography eluting with MeOH: CH2Cl2: trifluoroacetic acid (TFA) (8: 92: 0.001). The iso-A2E, which accounts for about 5% of total NMR, was isolated by HPLC Pure A2E was purified. To remove the TFA of the A2E-TFA salt, Dissolved in dichloromethane and washed with saturated sodium chloride solution. The obtained organic layer was washed with sodium sulfate Dried and concentrated under reduced pressure and used in the next step without further purification. BODIPY FL (Life technologies), which can be purchased as a product, was dissolved in THF (2 ml). DIC (N, N'-diisopropylcarbodiimide) and DMAP (4-dimethylaminopyridine) Was added and then the previously obtained A2E was added. The reaction was stirred for 24 h and TLC . The developing conditions were as follows: 10% dichloromethane in methanol, 0.2% TFA It was included condition. Finally, 4 mg of dark brown A2E-BDP was obtained. 97%
  • 81
  • [ 116-31-4 ]
  • C38H44P(1+)*Br(1-) [ No CAS ]
  • [ 13312-52-2 ]
YieldReaction ConditionsOperation in experiment
63% Stage #1: C38H44P(1+)*Br(1-) With n-butyllithium In tetrahydrofuran at -78℃; for 0.5h; Stage #2: all-trans-Retinal In tetrahydrofuran at -78 - 25℃; for 1.5h; 14 Step 14 1) After dissolving 0.86 g of phosphonium salt in 8 mL of THF, lower the temperature to -78 °C. 2) 0.88 mL of n-BuLi (1.6M) (the same number of moles as phosphonium salt) is slowly added dropwise and stirred for 30 minutes. 3) A solution of 0.39 g of all trans retinal dissolved in 50 mL of THF is added and stirred at -78 °C for 1 hour, followed by stirring at 25 °C for 30 minutes. 4) Add water, extract 3 times with E2O, and wash twice with brine. Remove residual moisture with Na2SO4. 5) The solvent is removed under reduced pressure and purified by silica column (C18-silica gel, CH3CN). 0.51 g (yield 63%) was obtained. This step is characterized by the reaction of linking phosphonium salt and aldehyde with carbon double bonds through the wittig reaction.
With potassium hydroxide In ethanol at 20 - 70℃; for 2.25h; Wittig reaction 9CBC formation (step g) (Goswami and Barua, 2003) (0081) [0060] As depicted in Scheme 1, step g, to a stirred solution of 86 mg of 9-cis retinyl triphenylphosphonium bromide (0.14 mmol) in 2 ml of dry ethanol at 70°C, all-trans retinal (19.7 mg, 0.069 mmol) in 17 ml of dry ethanol was added dropwise. KOH (0.43 g) dissolved in dry ethanol (5 ml) was added slowly to the mixture, and the solution was stirred at 70°C for 15 min and then at room temperature for 2 h. The reaction was monitored by TLC (8:2 hexane:ethyl acetate). When no more carotene formation was noted after 2 h, water (10 ml) was added to the reaction mixture and the product was extracted with portions of diethyl ether. The organic phase was removed under vacuum. MS (APPI) of crude cacld for C40H56 536.4, found 536.4 (M), 537.4 (M-H+). HPLC analysis of the crude (cosmosil cholester packed column 4.6mm I.D x250mm) isocratic elution of CH3OH / CHC13, 80:20 for 16 min (flow rate 2 mL/min, λ = 447 nm). Retention time was 11 min. (87% purity, Fig. 1). 9CBC purified by preparative HPLC (Cholester packed column) using elution of CH30H:CHC13, 85: 15 for 5 min, then 85: 15 to 80:20 for 5 min and then isocratic elution of 80:20 for 20 min (flow rate 25 mL/min, λ = 447 nm). Retention time was 16 min. MS (APPI) of crude cacld for C4oH56 536.4, found 536.4 (M), 537.4 (M-H+).
  • 82
  • [ 6541-41-9 ]
  • [ 116-31-4 ]
  • C10H14O2 [ No CAS ]
YieldReaction ConditionsOperation in experiment
With Fe(II) substituted apocarotenoid oxygenase from Synechocystis sp. PCC6803; oxygen In aq. buffer at 28℃; Enzymatic reaction; ACO activity assays The enzymatic activity of metal-free, native and putativemetal-substituted forms of ACO was measured by HPLC aspreviously described with minor changes [18]. Specifically,2 μg of purified ACO were added to a 200 μL reaction bufferconsisting of 20 mM HEPES-NaOH, pH 7 and 0.05% (w/v)Triton X-100 (Anatrace, Maumee, OH). Reactions wereinitiated by addition of an ethanolic solution of all-trans-8′-apocarotenal to a final concentration of 100 μM. Reactionmixtures were placed in a shaker-incubator operatingat 28 °C with 500 rpm shaking for 1 min and then quenchedby addition of 300 μL of methanol. The reaction products,all-trans-retinal and 8′-hydroxy-15′-apocarotenal, as wellas the remaining substrate were extracted with 500 μL ofhexane and analyzed directly by high-performance liquidchromatography as previously described [18]. The amountof generated all-trans-retinal was quantified by comparisonagainst a standard curve that was generated with knownamounts of all-trans-retinal (Toronto Research Chemicals,Toronto, Canada, > 95% purity). ACO activity was also measured spectrophotometricallyusing a Lambda Bio spectrometer (Perkin Elmer, Waltham,MA) or a Flexstation 3 plate reader (Molecular Devices, SanJose, CA) as previously described with minor changes [26,27]. Six microgram of purified ACO was added to 100 μL ofreaction buffer with or without test compounds. Following a10 min incubation period at 28 °C, all-trans-8′-apocarotenalwas added to the enzyme at a final concentration of 25 μMin a reaction volume of 200 μL. Assays were also performedwith protein omitted to determine the rate of non-enzymaticloss of apocarotenoid substrate.
  • 83
  • [ 116-31-4 ]
  • [ 95-54-5 ]
  • C26H34N2 [ No CAS ]
YieldReaction ConditionsOperation in experiment
87.5% In acetonitrile for 2h; Reflux; Synthesis of V1 The retinal (0.30 g, 1.06 mmol) and o-Phenylenediamine(0.34 g, 3.18 mmol) was refluxed in 35 mL acetonitrilfor 2 h. The solvent was evaporated by rotary evaporator, gettingcrud product. The crud product was recrystallized in 20 mL petroleumether and 2mL ethanol [24], cooling and vacuum filtration,obtaining orange solid 0.35 g, yield 87.5%. 1H NMR (400 MHz,DMSO-d6) d 8.64 (d, J 9.7 Hz, 1H), 7.08 (dd, J 8.0, 1.2 Hz, 1H),7.02e6.88 (m, 2H), 6.67 (dd, J 8.0, 1.3 Hz, 1H), 6.60e6.45 (m, 2H),6.44e6.33 (m, 1H), 6.24 (dt, J 29.7, 11.7 Hz, 3H), 5.06 (s, 2H), 2.20(s, 3H), 2.06e1.94 (m, 5H), 1.70 (s, 3H), 1.58 (dt, J 8.5, 4.7 Hz, 2H),1.49e1.39 (m, 2H), 1.02 (s, 6H). 13CNMR (101 MHz, CDCl3) d 155.25,154.42, 143.70, 137.63, 136.96, 135.45, 134.27, 131.43, 131.22, 129.85,124.84, 114.79, 112.57, 79.28, 39.65, 34.33, 33.29, 29.02, 21.87, 19.17,14.05, 13.18. HR-MS: calcd for C26H36N2 [MH], 375.2800; found:375.2803.
  • 84
  • [ 116-31-4 ]
  • [ 156-87-6 ]
  • N-retinylidene-N-retinylpropanolamine [ No CAS ]
YieldReaction ConditionsOperation in experiment
With acetic acid In ethanol at 20℃; for 48h; Inert atmosphere; Darkness; 4.4. Synthesis of A2E and A2P General procedure: Total lipids were extracted fromsamples by a modified Bligh and Dyer method [35,36]. Briefly, 1mgof RPE granules in suspension were mixed in 200 L H2O with a Bullet Blender (Next Advance).Methanol (200 L) was then added to the sample and mixed, followed by addition of 200 L ofchloroform (CHCl3) and mixing. The mixture was centrifuged at 14,000 rpm for 10 min in a tabletopmicrocentrifuge, and the lipid-containing lower phase was transferred to a collection tube. To ensurecomplete extraction, each sample was extracted 4 times with 200 L fresh chloroform added each time.Collected lipids in chloroform were pooled and dried in a SpeedVac (Savant Instruments), flushedwith argon, and stored at 20 C in the dark until use.The absolute amount of A2E in a sample was measured
  • 85
  • [ 116-31-4 ]
  • [ 141-43-5 ]
  • N-retinylidene-N-retinylethanolamine [ No CAS ]
YieldReaction ConditionsOperation in experiment
With acetic acid In ethanol at 20℃; for 48h; Inert atmosphere; Darkness; Enzymatic reaction; 4.4. Synthesis of A2E and A2P A2E was synthesized following a published method [34]. Ethanol (31.3 mL), ethanolamine(3.8 mL), acetic acid (4.5 mL), and all-trans-retinal (1 g) were added to a 50-mL tube and allowed toreact at room temperature in the dark for 2 days with gentle rocking. The ethanol was then evaporated,and the reaction mixture was dissolved in acetonitrile, washed 5 times with hexane and 1 M sodiumacetate (1:1). The middle layer was collected after each wash. The reaction product was washed onemore time with H2O and dried in a SpeedVac (Savant Instruments) overnight. Synthesized A2E wasstored under argon at 20 C in the dark.
  • 86
  • [ 41889-27-4 ]
  • [ 116-31-4 ]
YieldReaction ConditionsOperation in experiment
46% With pyridine; palladium diacetate; potassium carbonate In N,N-dimethyl-formamide at 60℃; for 6h; 1 Example 1: A 2-necked flask equipped with a stirring bar, thermometer and a dimrothcondenser was charged with K2C03 (16 mg, 0.15 eq), Pd(OAc)2 (16 mg), 11,12-dihydroretinal (190 mg, 1.0 eq), DMF (3.0 mL) and pyridine (5 iL, 0.1 eq). The yellow suspension was stirred at 60 °C for 6 h, applying a constant air stream. The reaction mixture was cooled to room temperature and diluted with Et20 (10 mL) and washed with H20 (10 mL x 3). The organic phase was concentrated underreduced pressure (40 °CI 30 mbar). Purification by column chromatography afforded the product as orange solid (85 mg, yield 46%).
  • 87
  • [ 75917-44-1 ]
  • [ 116-31-4 ]
YieldReaction ConditionsOperation in experiment
29% With 2,3,5,6-tetrafluoro-1,4-benzoquinone In toluene at 60℃; for 24h; 3 7.8-Dihydroretinal (150 mg, 1.0 eq) were dissolved in toluene (5 ml_) and fluoranil (2.0 eq) was added. The reaction mixture was stirred 24 h at 60°C. The solution was filtered over a plug of silica and all volatiles were evaporated under reduced pressure. Purification by column chromatography afforded the desired product (29% yield).
  • 88
  • [ 116-31-4 ]
  • cellular retinol-binding protein-1 [ No CAS ]
  • cellular retinol-binding protein-1 in complex with all-trans retinaldehyde [ No CAS ]
YieldReaction ConditionsOperation in experiment
With sodium chloride In aq. phosphate buffer for 0.333333h; Cooling with ice; Preparation of holo-CRBPs General procedure: Holo-CRBPs were prepared immediately before assays by incubating apo-CRBPs with a 2.5-fold molar excess of retinoid for 30 min on ice (<2% ethanol final, v/v) in 50 mM potassium phosphate (pH 7.4) buffer containing 25 mM NaCl. The CRBP-retinoid solution was then centrifuged at 25,000g for 20 min at 4 °C. The supernatant was applied to a 5 ml HiTrap Desalting column with a syringe, according to manufacturer’s instructions. Absorbance spectra of purified holo-CRBPs were recorded from 220 to 500 nm to ensure retinoid binding (representative spectra in Fig. S2). The concentration of each holo-CRBP was calculated using previously determined extinction coefficients (see Table S1).
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