Amino ninhydrin coupling

The specific detection of aromatic nitro compounds is a second example. These can be converted by reduction to primary amines, which are then diazotized and coupled to yield azo dyes (cf. reagent sequence Titanium(III) chloride — Bratton-Marshall reagent ). Sodium nitrite —naphthol reagent, diazotized sulfanilic acid and other reagents specific for amino groups (e.g. ninhydrin, fluorescamine, DOOB, NBD chloride [9]) can also be used in the second stage of the reaction (Fig. 21). 

There are no special requirements in the selection of an N-terminal amino acid residue in a segment with which the carboxy component of a segment is to be coupled, unless a highly hindered amino acid or a secondary amino acid is selected. If a Pro or Hyp residue is located at the N-terminus of the segment, monitoring of the coupling reaction with ninhydrin or fluorescamine is extremely difficult. 

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. 

Fmoc-Rink-Nle-pMeBHA-resin (1 g, 0.55 mmol g ) was swollen for 1.5 h in NMP in a reaction vessel equipped with a sintered glass bottom, and placed on a shaker. The Fmoc group was removed with 20% piperidine in NMP (8mL, 2 x 15 min) and after washing with NMP (8mL, 5 x 2 min), the Fmoc removal was monitored by the ninhydrin Kaiser test. Coupling of building blocks such as iV -Fmoc-/Va>-carb-oxyalkyl(OAl)Xaa-OH or /V -Fmoc-/V" -aminoalkyl(Aloc)Xaa-OH, or N -alkylated amino acids such as... 

The microwave protocol increased the reaction rate at least two- to threefold, as conversion was only 60-80% within 6 min under conventional heating. This improved coupling efficiency was duplicated with numerous amino acid derivatives and a further two peptide fragments were coupled with the Gly-Wang resin. These couplings were completed within 2 min as determined by quantitative ninhydrin assay. 

The Kaiser test uses three solutions ninhydrin (5 g in 100 ml of ethanol), phenol (80 g crystalline phenol in 20 ml ethanol), and pyridine/KCN [dilute 2 ml of 0.001 mol/liter aqueous solution of KCN (potassium cyanide) with 98 ml of pyridine]. The test is performed by transferring a few resin beads to a 6 x 50-mm glass test tube. The beads are washed with ethanol and a drop of each of the three testing solutions is added. The tube is then heated to 100° for 5 min in a heating block. Blue (brown for proline)-colored beads and solution (positive test) indicate the presence of free amino groups on the resin. Colorless beads (negative test) indicate complete coupling. The quality of phenol is important. Impure phenol may result in a false-positive Kaiser test. 

Peptide carbothioic acids were synthesized using a thioacid linker 4 developed by Blake (Scheme 4).P - l A generalized synthesis of this Boc-Xaa-thioacid-linker has been described by Canne et The thioadd-resin was synthesized by dissolving Boc-Xaa-thioacid-linker DCH A salt (1.1 equiv) and HBTU (1.1 equiv) in a minimum volume of DMF and activated for 2 min. This mixture was added to amino-methyl-polystyrene-resin (1.0 equiv, -l.Ommol-g loading of amine) and coupled for Ih or until the reaction proceeded to >9 5% as monitored by the quantitative ninhydrin test. 

The synthesizers described in this section and listed in Table 5 are capable of containing 100 g or more of resin.f l The approach in a large-scale chain elongation is different from research scale methods. For example, a 0.25 mmol synthesis may use 4 to 5 equivalents of amino acid and couple for 30 minutes. By contrast, a 250 mmol synthesis may implement only 1.5 to 2.5 equivalents of amino acid and couple for 90 minutes or longer. Resin samples are usually removed after every coupling and the ninhydrin test is performed to verify reaction completion prior to starting the next cycle (for ninhydrin monitoring see Section 4.3.6.5.1). 

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