Ninhydrin test

Further development of the chromatogram with 0.1N ammonia afforded fractions positive to ninhydrin test. From the fractions, 249 mg. of a colorless material was obtained. It was dissolved in 19 ml. of water and the solution was adjusted to pH 4.0 with dilute hydrochloric acid. A colorless material, after condensing under a reduced pressure and lyophilization, was recrystallized from aqueous methanol with a small amount... 

Ninhydrin Assays. Ninhydrin tests were performed using a modified procedme of Taylor et al. " APS Silica (10-75 mg) of various loadings (0.857, 0.571, and 0.343 mmol NH2/g Silica) was added to phosphate buffer (5 mL, 100 mM, pH 6.5), and 1 mL of a 5% w/v solution of ninhydrin in ethanol was added to the sluny. After stirring for an hour in a boiling water bath, the mixture was allowed to cool slowly to room temperature. The silica was then filtered and washed three times with 70°C distilled water. The filtrate was collected, added to a volumetric flask, diluted to 100 mL, and the absorbance of this solution at 565 mu was measured using a UV-visible spectrophotometer. The reference solution was prepared as above with unmodified amine-free silica. Calibration standards were prepared with aliquots of a 1 mg/mL solution of APS in ethanol. 

Figure 38.1. Ninhydrin tests on grafted APS silica materials. The two samples with 0.857 mmol/g loadings were tested using different solutions on different days to test reproducibility from day-to-day.

Normally the reaction Is useful for the conversion of alkyl halides to primary amines without concomitant formation of secondary amines.29 Treatment of polymer 17 with hexamethylenetetramine in a mixture of ethanol/THF afforded an insoluble resin. Using diazabicyclooctane (DABCO), we demonstrated that the reaction could be limited to attack by a single nitrogen in a multifunctional amine, so we did not anticipate crosslinking via bis-quat salt formation. Hydrolysis of 2 with anhydrous HC1 in ethanol generated free amino groups as evidenced by a positive ninhydrin test, but quantitative hydrolysis could not be achieved and the product remained insoluble. One would have expected a simple bis-quat to hydrolyse and open the crosslinked structure. 

The ninhydrin test was performed according to a published procedure.2 Potassium cyanide/pyridine (0.0002M), phenol/ ethanol (76% w/w), and ninhydrin/ethanol (0.28 M) were purchased from Perkin-Elmer. 

FIGURE 5.5 Schedule for the solid-phase synthesis of somatostatin, a 14-mer, on 10 g of resin reacted with 5 mequiv of the first amino acid, adapted from J. Rivier, J. Am. Chem. Soc. 96 2986, 1974. Min = time of mixing X2 = two times DCC = dicyclohexylcarbodiimide. Step 4 included 5% of (CH2SH)2 to prevent the oxidation of tryptophan. When the ninhydrin test on an aliquot after step 13 was negative, step 1 followed when positive, steps 9-13 were repeated. 

Attachment of Compound 11 onto an Aminomethylated Polystyrene Resin 1 3141 A 1% cross-linked aminomethylated polystyrene resin (0.83 mmol of amino group per gram of resin, 2.41 g, 2 mmol) was placed in an ATC Model 90 reaction vessel (capacity 200 mL) and the resin washed successively with NMP, 10% DIPEA in NMP, and NMP. Then, 11 (1.41 g, 3 mmol, 1.5 equiv), HBTU (1.14 g, 3 mmol), and HOBt (0.41 g, 3 mmol) were dissolved in NMP (20 mL) in a flask, and DIPEA (0.78 mL, 4.5 mmol) was added to the soln immediately. The mixture was introduced into the reaction vessel containing the resin within 2 min after addition of DIPEA, and the entire mixture was agitated for lh at rt. The soln was drained from the reaction vessel and the resin washed well with NMP. The completion of the coupling reaction was confirmed by a ninhydrin test. 

On confirming that the ninhydrin test is negative, the resin is subjected to the segment elongation reaction. If the ninhydrin test is positive, the same coupling procedure should be repeated until the ninhydrin test becomes negative. 

DIPEA (8 8 12 mmol each) as the coupling reagents. Completion of each coupling reaction was monitored by the ninhydrin test. Finally, the fully protected peptide-HMFS-resin was collected by filtration on a glass filter, washed well with MeOH and dried wt of the resin 5.97 g. 

The peptide resin 1 was treated for 1 h with 50% TFA in CH2C12 containing 2% anisole, washed with CH2C12, neutralized with 5% DIPEA in CH2C12, washed with CH2C12, and then DMF. The product 2 was treated with a 5-fold molar excess of HOBt in DMF, followed after 20 min with a 5-fold molar excess of DCC in CH2C12. The mixture was adjusted with DIPEA to pH 7.5. After 24 h, the resin was drained and after a neutralization step with DIPEA, the cyclization was repeated for another 24 h. The extent of the cyclization to 3 was estimated to be about 95% by the ninhydrin test. 

To perform the ninhydrin test a solution of ninhydrin in ethanol or acetone (0.2% w/v) is sprayed lightly over the plate. Brown to purple colors develop slowly (within 1-2 min) upon heating to 60-90 °C. The spray is stable for several weeks. 

In order to prepare the Pauly reagent two solutions, solution I (0.4 M sodium sulfanilate in water) and solution II (0.4 M sodium nitrite in water) are made separately. Just before use, solution I with solution II, 0.25 M HC1, and 2 M NaOH (1 1 8 10) are mixed successively. This solution, which is not stable, should be applied as a spray within minutes and discarded. The ninhydrin test will not interfere with the Pauly reagent. 

The ninhydrin test will not interfere with the Ehrlich reagent. Therefore, a plate can be examined under UV light, immersed in iodine vapors, treated with ninhydrin, and the Pauly or Ehrlich reagents successively. 

The Ninhydrin test is useful for monitoring the reaction see Note 5). 

After washing with acetonitrile (1 mL, 6 times), the resins were washed with a saturated aqueous solution of sodium bicarbonate (200 pL), and then washed with water (more than 6 times) see Note 10). The Ninhydrin test is useful for estimation of the reaction ration. 

For Ninhydrin test, small amount of the sample resins was heated in a mixture of 25 mL of 0.28 M Ninhydrin/EtOH solution and 100 mL of pyridine at 100°C for 10 min. The same amount of unused Toyopearl was used for the control. The reaction ratio was estimated based on the OD570 absorption value. 

In a subsequent paper on the use of the Stille reaction to form biaryls,58 the reaction was carried out under ambient conditions to allow robotic automation of the process. Attachment of the tin species to resin was very straightforward, in that 4-tri-n-butylstannylphenyl acetic acid was linked to Rink amide resin simply using a DIC coupling. Loading of this species was determined by tin elemental analysis and correlated with a quantitative ninhydrin test of free amines remaining on the support (Scheme 24). 

After the reaction times given in Scheme 38, the respective couplings were quantitative according to chloranil and the Kaiser ninhydrin test and/or HPLC of small cleavage. The peak area of the crude Fmoc-deprotected dodecapeptide was 90% (HPLC k=214nm). The pure cyclic peptide was obtained in an overall yield of 31%. Cyclosporin O was synthesized by a similar procedure.129 ... 

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