Cholest-4-en-3-one (Aldrich *) is dissolved in 100 mL 2-propanol-10% H₂O with warming. A 1.0 mL sample is taken as a standard. NaB(OAc)₃H (Aldrich **) is added. A white ppt. forms almost immediately.
After 24h reaction, TLC (silica gel, AcOEt:hexane::1:9) shows that the reduction to α,β-allocholestanol (R_F, **, **) is complete. The starting material reacts strongly with the PMA and in UV+.The product reacts only weakly with PMA and is UV-. 100 mL H₂O is added to the reaction mixture and it is cooled to ~10° in the refrigerator.
The white precipitate is removed by vacuum filtration, the precipitate washed well with water and dried to constant weight over P₂O₅.
The product is resolved into the α and β-forms by flash chromatography on an ISCO Companion on silica gel using a hexane eluant with an acetone gradient (blue).

1.04g (1.96mmol, 2X¹) of Fmoc-Phe-OPfp (Bachem B-1170) was dissolved in 5 mL DMA with vortex mixing and added to 20 mL of a DMA solution of Gly-OaChl (0.982mmol). The rate of coupling was followed by the standard assay². The coupling reaction appeared to have stopped after 30s.
At 5m an additional 0.25g Fmoc-Phe-OPfp (0.47mmol) was added to the reaction mixture leading to a final A₅₇₀ of 0.016³.
After two hours of additional reaction the solution was made 20% (v/v) in piperidine and incubated 20m at room temperature to remove the Fmoc-group. Volume of the reaction mixture is reduced to ~5mL by rotary evaporating at high vacuum and the mixture containing the dipeptide chromatographed on LH-20 Sephadex (95 x 1.5cm) with a DMA eluant. The first 80 mL of the eluant was collected as a unit and 2.0 mL fractions taken subsequently. 10μL aliquots of the fractions were checked by reaction with ninhydrin to give the elution profile shown on the right. Fractions 6-14 (93-116g) were pooled.
Independent studies show that side products such as Phe-Pip, piperidine, HOPfp elute ≥130 g DMA. R for the chromatography is ** indicating that the lead peak contains less than 0.**% ninhydrin positive impurities⁴.

1.07g ( 1.89 mmol, 2X) of Fmoc-Glu(OtBu)-OPfp (Bachem B-1130) was dissolved in 5 mL DMA and added to 20 mL of a DMA solution of Gly-OaChl. The rate of peptide bond formation is shown on the right. Half-time for the coupling reaction is less than 30s.
After two hours the reaction mixture is made 20% in piperidine and incubated 20m to remove the Fmoc group. Volume of the reaction mixture is reduced to ~5mL and the dipeptide chromatographed on LH-20 Sephadex in DMA as shown on the right. The first 70 mL of the eluant was collected as a unit and 2.0 mL fractions taken subsequently. R for the chromatography is ** indicating that the lead peak contains less than 0.**% ninhydrin positive impurities. Fractions eluting between 77 – 106 mL are pooled to yield the tripeptide.

0.88g ( 1.74mmol, 2X) of Fmoc-Val-OPfp (Bachem B-1565) are dissolved in 5 mL DMA and added to DMA solution of the tripeptide. The rate of coupling is plotted in the Figure on the right. T1/2 for the coupling reaction is ~3m.
Removal of the Fmoc group was accomplished as before, volume of the reaction mixture reduced to ~5mL and the crude tetrapeptide reaction mixture chromatographed on LH-20 Sephadex in DMA. The first 80 mL of the eluant was collected as a unit and 2.0 mL fractions taken subsequently. Fractions eluting between 79 – 104 mL are pooled to yield a solution of homogeneous tetrapeptide. The elution profile is shown on the right.

0.89g (1.00mmol, 2X) of Fmoc-Gln-OPfp (Bachem B-1125) is dissolved in 5 mL DMA and added to the pooled fractions containing the tetrapeptide. DMA solution of Gly-OaChl. The rate of coupling is shown on the right. Half-time for the coupling reaction is ~1m. After two hours of reaction the Fmoc-group is removed with piperidine as described previously, volume of the reaction mixture reduced to ~5mL and the pentapeptide chromatographed on LH-20 Sephadex in DMA. Fractions eluting between 76 – 102 mL are pooled to yield the pentapeptide. R for the chromatography is ** indicating that the lead peak contains less than 0.**% ninhydrin positive impurities.

0.76g (1.60mmol, 2X) of Fmoc-Ala-OPfp (Bachem B-1100) is dissolved in 5 mL DMA, added to the pooled fractions containing the pentapeptide and the coupling reaction progress followed by analysis with ninhydrin. The coupling reaction is complete within 0.50m (Figure on right). The Fmoc-group is removed with piperidine as described previously and the hexapeptide purified by chromatography on LH-20 Sephadex in DMA. R for the separation is > 2.0.
Fractions eluting between 75 – 102 mL are pooled to yield purified hexapeptide.

10 μL aliquots of the pool chromatographic fractions are diluted with a 79.8: 20.0:0.2::methanol:water:formic acid mixture to give a solution that is approximately 10μM. This is then serially diluted with the same solvent to give mixtures containing between 0.03 - 10μM. Infusion of these mixtures into a * at 5μL/minute.
In the initial scan between 300 – 2000 AMU is made to identify the molecular ion.
Counts for the molecular ion ±2 are collected in SIM mode for the various concentrations for 3 – 1minute intervals, averaged, and the background subtracted to give the linear plot shown on the right.
The equation obtained by a best fit is then used to quantitate the amount of uncoupled tetrapeptide in the solu-tion of pentapeptide, etc. Because of its high sensitivity (<0.05% uncoupled material), tiny amounts of product required, and specificity this assay is a reasonable way to measure uncoupled material in the product.
The sensitivity of the technique varies with the molecular weight but typically <100nM uncoupled material can be detected in a 10 mM solution of reaction mixture.
Analysis indicates that at various steps in the synthesis of the crude hexapeptide, the total amount of uncoupled amino component that could give rise to deletion sequences contains <0.12 percent uncoupled material.