Fmoc-protected amino acids

Investigation of the microwave-assisted attachment of Fmoc-protected amino acids onto 2-chlorotrityl chloride resin indicated higher loadings and increased rates compared to standard room temperature procedures [146]. In this comparative study standard procedures yielded 0.37 mmol/g loading after 1 hour, whereas at 110 °C using microwave dielectric heating, a similar result (0.38 mmol/g) was obtained after only 15 min (Fig. 7). 

The earliest published example of microwave-assisted SPOS involved diisopropyl-carbodiimide (DlC)-mediated solid-phase peptide couplings [24], Numerous Fmoc-protected amino acids and peptide fragments were coupled with glycine-preloaded polystyrene Wang resin (PS-Wang) in DMF, using either the symmetric anhydride or preformed N-hydroxybenzotriazole active esters (HOBt) as precursors (Scheme 12.1). 

A total of 36 MicroTubes ( 42 pmol / MicroTube) were sorted into three vials (note 9). MicroTubes in each vial were treated at room temperature with Fmoc-protected amino acids (2,5.4 mmol, 10 equiv note 10), DIEA (1.75 mL, 10.08 mmol, 20 Eq.), and HATU (1.91 g, 5.04mmol, 10 Eq.) in DCM (24mL) for 24h. After the supernatant was removed by aspiration, the MicroTubes were then washed three times with DMF, DCM, MeOH, and DCM. The MicroTubes were dried under vacuum overnight. IR 1657 cm-1 (CONHR note 11). 

Sequential acylation reactions were carried out at ambient temperature for 1.5 h using a DMF solution (1.3 mL) of the appropriate A-Fmoc-protected amino acids [Fmoc-Arg/D-Arg(Pmc)-OH, 265 mg Fmoc-Trp/D-Trp(Boc)-OH, 211 mg 0.4 mmol) and then carboxyl activated using TBTU (154 mg, 0.4mmol), HOBt (54mg, 0.4mmol), and DIEA (140pL, 0.8 mmol). Repetitive A"-Fmoc deprotection was achieved using 20% v/v piperidine in DMF (6 min, 2.5 mL min - ). 

M Green, J Berman. Preparation of pentafluorophenyl esters of Fmoc protected amino acids with pentafluorophenyl trifluoroacetate. Tetrahedron Lett 31, 5851, 1990. 

While the distinct amino acid residues have mostly only a modulating effect (see Table 1.9) (e.g., FMOC-protected amino acids), the type of protection group or derivative formed decides on the molecular and chiral recognition mechanism and hence on the obtained elution order as well as the level of enantiomer recognition (i.e., magnitudes of a-values) that can be afforded. From a practical point of view, we may distinguish between two groups of IV-derivatives ... 

TFA-induced cleavage in the course of which the dendrimer core remained intact. 2-Naphtalenesulfonamide (57) was obtained in 84% yield. Compound (58) was obtained via support-deprotection and immobilization of a Fmoc protected amino acid by means of HATU coupling. The supported substrate was deprotected and acylated with phenylacetic acid using standard coupling protocols. The final step was the product cleavage, which delivered the phenylalaninamide (58) in 87% yield. 

Although the simplest route to prepare hydroxamic acid derivatives remains the reaction of hydroxylamine with acid chlorides, this last method cannot be apphed to all Af-protected-a-amino acids. The synthesis of Fmoc-protected amino acid hydroxamates represents the only exception to this rule . In fact, Fmoc-amino acid hydroxamates 98 can be synthesized by the acylation of hydroxylamine using Fmoc-amino acid chlorides 97 in the presence of MgO (Scheme 52). The route is simple, efficient, and affords good yields of products. 

Piperazine-2,5-diones can be symmetric or asymmetric. Symmetric DKPs are readily obtained by heating amino acid esters,1179-181 whereas asymmetric DKPs are obtained directly from the related dipeptides under basic or, more properly, acid catalysis, or by cyclocondensation of dipeptide esters.1182-185 As an alternative procedure hexafluoroacetone can be used to protect/activate the amino acid for the synthesis of symmetric DKPs or of the second amino acid residue for synthesis of the dipeptide ester and subsequent direct cyclocondensation to DKPs.1186 The use of active esters for the cyclocondensation is less appropriate since it may lead to epimerization when a chiral amino acid is involved as the carboxy component in the cyclization reaction. Resin-bound DKPs as scaffolds for further on-resin transformations are readily prepared using the backbone amide linker (BAL) approach, where the amino acid ester is attached to the BAL resin by its a-amino group and then acylated with a Fmoc-protected amino acid by the HATU procedure, N -deprotection leads to on-resin DKP formation1172 (see Section 6.8.3.2.2.3). 

H-Thz-OH (1.33 g, 10 mmol) was dissolved in dry DMF (100 mL) in the presence of DIPEA (3.23 mL, 19 mmol). A soln of Fmoc-protected amino acid fluoride (9 mmol) in dry DMF (100 mL) was added dropwise, and the mixture stirred for 2 h at rt. The soln was then concentrated, the residue taken up in EtOAc, washed with aq 5% citric acid and brine, and dried (Na2S04). The solvent was removed and the residue purified by flash chromatography (silica gel, EtOAc/AcOH/petroleum ether) yields 60-95%. 

The peptide was synthesized on Rink resin (0.2 mmol) using Pfp activated ester of the Fmoc-protected amino acids. All couplings were complete after 2 h. After acetylation, the peptide was cleaved by a 2 min treatment with a 50% TFA soln in CH2C12. After filtration, the TFA soln was concentrated by a stream of N2 to 1-2 mL and the crude peptide was precipitated by adding Et20 (25 mL) and cooling to —78 °C. The Et20 layer was removed and the peptide dried under vacuum. After purification by RP-HPLC pure peptide 53 was obtained yield 101 mg (36%) HRFAB-MS mlz 1408.55180 [M + H]+. 

The above compound 66 (1.22 g, 2.51 mmol) was dissolved in anhyd acetone (20 mL) and cooled to 0°C. The soln was saturated with anhyd HC1 and stirred at rt for 30 min. After the solvent was removed, the dihydrochloride 67 was neutralized with 10% Na2C03 and washed with hexane. A soln of Fmoc-OSu (931 mg, 2.51 mmol) in dioxane (26 mL) was added dropwise to the amino acid soln at 5°C and the mixture was stirred at rt for 12 h. The mixture was diluted with H20 and washed with Et20. The aqueous phase was acidified to pH 3 with coned HC1 and the suspension formed extracted with EtOAc. The organic phase was dried (Na2S04) and concentrated to yield the desired Fmoc-protected amino acid 60 yield 1.11 g (91% yield for the two steps) TLC Rf 0.14 (silica gel, CHCI3/MeOH 5 1) [a]D25 +18.8 (c 0.5, CHC13) HRFAB-MS mlz 489.2014 [M + H]+. 

The reagents sketched in Figure 13.2 are stable and can be prepared either in solution or on insoluble supports. Activated Boc- or Fmoc-protected amino acid derivatives that are sufficiently stable to be isolated, some of which are commercially available, include acyl chlorides [9,13], fluorides [10,14,15], symmetric anhydrides [16], pentafluorophenyl esters, Af-hydroxysuccinimidyl esters, and 4-nitrophenyl esters [17,18],... 

During acylations with Fmoc-protected amino acids, addition of bases should be avoided as these could lead to partial deprotection and thence to multiple incorporation of the amino acid. Small amounts of DIPEA or pyridine, however, do not usually cause major problems (see Experimental Procedure 13.5). 

The synthesis was carried out using 125 Chiron Mimotopes Crowns (capacity 5.3 pmol each) derivatized with an Fmoc -Rink amide linker. The procedure was started with the formation of five strings by threading 25 crown units on Berkley Fire Line fishing line. Five Fmoc-protected amino acids were used in each coupling position as demonstrated in the flow diagram of the synthesis (Fig. 11). 

A solution of the amino acid (0.40 mmol) and potassium carbonate (39 mg, 0.40 mmol) in water (15 ml) was added to a suspension of Fmoc-P-OSu (270 mg, 0.40 mmol) in acetone (20 ml). The suspension was stirred at room temperature for 24 h and the solvents were removed under reduced pressure. Toluene was added (20 ml) and the resulting solid was filtered. The solid was suspended in water (20 ml) and the P-HOSu (A-hydroxysuccinimide) was removed by filtration. The filtrate was acidified with concentrated HC1 (2 ml) and extracted with ethyl acetate (3 x 20 ml). The combined organic layers were dried over sodium sulfate and evaporated to give the pure Fmoc-protected amino acids. 

Limitations in product size were investigated by preparing several peptides of various lengths. Peptides containing 7, 9,13, and 19 amino acid residues were prepared on a synthesis robot employing Fmoc-protected amino acids and carbodiimide-hydroxybenzotriazol activation (0.25 M, 1 h) without detection of deletion products. To illustrate the remarkable economy of the ULTRA resin, 3.4 mg of the starting resin 15 sufficed for the synthesis of 42 mg resin with fully protected tridecapeptide. The raw product was obtained by ethereal trituration in excellent purity and yield (90% purity in the raw product, 78% yield, 13.1 mg after preparative HPLC). 

One method involves the use of preactivated Fmoc-protected amino acids (e.g., pentafluorophenyl ester). This method has the advantage that only one reagent is necessary since preparation of the amino acid solutions is very simple and the likelihood of mistakes is low. One disadvantage is the higher price of the amino acid derivatives, but due to the small amount of activated amino acids used the absolute difference falls in the range of a few dollars for synthesis of an entire peptide membrane array. Another disadvantage lies in the fact that activated esters are only commercially available for the standard amino acids. 

The other method requires the use of in situ activated amino acids. Activation of the amino acids is carried out by adding an activator and coupling reagent to the unactivated Fmoc-protected amino acid derivative. This method is more time... 

Glycosyl acceptors Fmoc-protected amino acids were obtained from Novabiochem D/L-hydroxylysine dihydrochloride monohydrate was obtained from Acros Organics. 

Standard Fmoc-protected amino acids are purchased from Advanced Chemtech (see Note 4). Unusual amino acids (all from Bachem) ... 

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