FMOC derivatives

The Bsmoc derivative is formed from the chloroformate or the A -hydroxy-succinimide ester. It is cleaved rapidly by a Michael addition with tris(2-aminoethyl)amine at a rate that leaves Fmoc derivatives intact. More hindered bases, such as A -methylcyclohexylamine or diisopropylamine, do not react with the Bsmoc group, but do cleave the Fmoc group, illustrating the importance of steric effects in additions to Michael acceptors. 

H2, Pd black, EtOH, 45°, 92% yield.If the hydrogenolysis is performed in the presence of (B0C)20 or Fmoc-OSu, the released amine is converted to the BOC and Fmoc derivatives in situ ... 

Fmoc derivative (Section 26.7) A fluorenylmethyloxy-carbonyl amide-protected amino acid. 

Takahashi and coworkers described an effective sialylation method utilizing the N-Fmoc, N-Troc and N-trichloroacetyl-P-thiophenyl sialosides (Scheme 4.6d) [167]. It was found that the N-Troc derivative of N-acetylneuraminic acid performed better than the corresponding N-Fmoc derivative. An N-Troc P-thiosialoside was applied for the synthesis of glycosyl amino acids by one-pot glycosylation [167]. Importantly, it was found that the N-Troc protecting group could be converted into an acetamido moiety without causing racemization of the peptide. 

The resin substitution level is based on spectrophotometric determination of the Fmoc-derived chromophore liberated upon treatment with 20% piperidine/DMF using e29o nm = 5253 M 1 cm-1, which was used to calculate the percent efficiency. 

To accurately determine anchoring, coupling, and cleavage yields, resins are extended further with an internal reference amino acid2 (IRAA lie is used), introduced as its Fmoc derivative by standard coupling methods, at a point before introduction of the handle. 

FIGURE 1.30 Micro-HPLC separation of all 4 stereoisomers of the dipeptide alanyl-alanine as FMOC derivatives (a) and DNP-derivatives (b), respectively, on a 0-9-(tert-butylcarbamoyl)quinine-based CSP. Experimental conditions Column dimension, 150 X 0.5 mm ID mobile phase (a) acetonitrile-methanol (80 20 v/v) containing 400 mM acetic acid and 4 mM triethylamine, and (b) methanol-0.5 M ammonium acetate buffer (80 20 v/v) (pHa 6.0) flow rate, 10 ixLmin temperature, 25 C injection volume, 250 nL detection, UV at 250 nm. (Reproduced fromC. Czerwenka et al., J. Pharm. Biomed. Anal., 30 1789 (2003). With permission.)... 

In addition, peak VI (fig. 1) contained two compounds, one identified as lysinoalanine (table 1). Lysinoalanine is a well-known artefact of alkaline protein treatment but is supposed to be formed in dentin by the reaction between a collagen lysine- and a phosphoprotein phosphoserine residue (Fujimoto et al., 1981). Both compounds were not detected by HPLC after FMOC-derivatization, most likely because of fluorescence quenching inherent to the close vicinity of several FMOC groups attached to one molecule. Thus the unknown compound seems rather similar to lysinoalanine. We suggest the unknown compound is histidinoalanine, which is present in dentin (Fujimoto et al., 1982) and likely shows fluo-rence quenching in its FMOC derivate. 

Hi. Lysine. Gamma radiolysis of aerated aqueous solution of lysine (94) has been shown, as inferred from iodometric measurements, to give rise to hydroperoxides in a similar yield to that observed for valine and leucine. However, attempts to isolate by HPLC the peroxidic derivatives using the post-column derivatization chemiluminescence detection approach were unsuccessful. This was assumed to be due to the instability of the lysine hydroperoxides under the conditions of HPLC analysis. Indirect evidence for the OH-mediated formation of hydroperoxides was provided by the isolation of four hydroxylated derivatives of lysine as 9-fluoromethyl chloroformate (FMOC) derivatives . Interestingly, NaBILj reduction of the irradiated lysine solutions before FMOC derivatization is accompanied by a notable increase in the yields of hydroxylysine isomers. Among the latter oxidized compounds, 3-hydroxy lysine was characterized by extensive H NMR and ESI-MS measurements whereas one diastereomer of 4-hydroxylysine and the two isomeric forms of 5-hydroxylysine were identified by comparison of their HPLC features as FMOC derivatives with those of authentic samples prepared by chemical synthesis. A reasonable mechanism for the formation of the four different hydroxylysines and, therefore, of related hydroperoxides 98-100, involves initial OH-mediated hydrogen abstraction followed by O2 addition to the carbon-centered radicals 95-97 thus formed and subsequent reduction of the resulting peroxyl radicals (equation 55). 

Following cleavage with hydrogen fluoride, the various classes of peptides were separated in a one-step purification procedure on a tertiary or quaternary amine column. After removal of the Sulfmoc group with 5% TEA, homogeneous Leu-Ala-Gly-Val, for example, was obtained. The Sulfmoc procedure was also very effective for purification of synthetic thymosin oq (28 residues). 87 This was the first use of an Fmoc derivative for selective and reversible orthogonal peptide purification. 

The Se-(4-methoxybenzyl)selenocysteine is obtained by reduction of selenocystine with NaBH4 and in situ reaction with 4-methoxybenzyl chloride. 7 The optimized procedure of Tanaka and Soda 32 is preferentially used for the synthesis of the starting selenocystine, which involves reaction of (1-chloroalanine with a 2.3-fold excess of disodium diselenide in aqueous solution at pH 9. Alternatively, the significantly less selenium demanding synthesis of Stocking et al. 33 is used for the preparation of expensive 77Se-selenocystine, this consists of the reaction of methyl (2R)-2-[(/ert-butoxycarbonyl)amino]-3-iodopropanoate with equivalent amounts of dilithium diselenide. Subsequent conversion of SeC(Mob) into the M -Fmoc derivative 7 and finally into the pentafluorophenyl ester 10 is performed following standard procedures. 

The tetra-O-benzylated A-Fmoc derivative of 165 was then incorporated by solid-phase synthesis into a helix-forming glycoheptadecapeptide, Ac-Tyr-Lys-Ala-Ala-Ala-Ala-Lys-Ala-Ala-Cgaa((3-D-Gal-C-)-Ala-Lys-Ala-Ala-Ala-Ala-Lys-NH2 (Cgaa= C-glycosylated amino acid)J128l... 

The Na-Z- or 7V"-Boc-protected lipo-amino acids and their esters are obtained by standard protocols.113X In contrast, for the synthesis of the A -Fmoc derivatives intermediate bis-silylation and the use of Fmoc-Cl as acylating agent are recommended. 129 The standard procedure with Fmoc-OSu leads to low yields (25%) mainly due to low solubility of the amino acids. 133 Alternatively, the improved solubility of the 4-toluenesulfonate salt of the benzyl esters is exploited for reaction with Fmoc-OSu. The benzyl ester is then removed by catalytic hydrogenation, although the Fmoc group is known to be reduced under these conditions unless particular precautions are taken. 133 156-159 ... 

To allow for the use of the stronger sulfating reagent pyridine/S03, an alternative protection strategy has been proposed by Fujii et al. 55-99 that is based on postsynthetic protection of the amino groups as Fmoc derivatives and of the serine hydroxy groups as terf-butyldiphenylsilyl derivatives as shown in Scheme 12. The histidine side chain is not protected since model studies indicate that sulfated histidine is readily hydrolyzed by water within 60 minutes. 

For the direct use of tyrosine 0-sulfate in peptide synthesis a set of -protected Tyr(S03H) derivatives were synthesized that can be combined with the most common protection schemes. In addition to Z-Tyr(S03Ba1/2)-0Ba1/2 (11)[3S1 (see Section 6.6.1.1.4), the related Boc and Fmoc derivatives have been synthesised as barium, sodium, potassium salts, and even as tetrabutylammonium salts due to their better solubility in solvents generally used for automated synthesis on solid supports.1152 154 155 ... 

Among the methods described for the synthesis of the key intermediate 4-(chloromethyl)-L-phenylalaninej123-125 direct chloromethylation of a protected l-phenylalanine followed by deprotection represents the simplest procedure. 126 This intermediate is converted into the 4-sulfomethyl derivative which is then used to prepare the /V -/ert-butyloxycarbonyl-4-(sulfo-methyl)phenylalanine monosodium salt l2S and the related Fmoc derivative. 126 ... 

The related Fmoc derivative was obtained by cleavage of the Boc group with 50% TFA in CH2C12 and reaction with Fmoc-OSu in aq Na2C03/THF under standard conditions yield 1.9 g (100%). 

A-(tert-Butoxycarbonyl)azetidine-2-carboxylic acid is obtained by reaction of azetidine-2-carboxylic acid with Boc-N3, m 112 Boc20, 113-"4 or 2-(fert-butoxycarbonyloxyimino)-2-phe-nylacetonitrile. 115 In a similar manner, the Fmoc derivative is prepared by the standard procedure using Fmoc-OSu as acylating agent.1"6 For derivatives of azetidine-2-carboxylic acid, see Table 4. 

Na-Protection of pipecolic acid (6) as its Boc derivative is carried out by standard procedures with Boc-N3, 177 208 B0C2O, 113114 or with 2-(/< rt-butoxycarbonyloxyimino)-2-phenylacetoni-trile. 235 Similarly, the Fmoc derivative is prepared in a standard manner (Table 9). 2 1 207 ... 

The development of chiral peptide-based metal catalysts has also been studied. The group of Gilbertson has synthesized several phosphine-modified amino adds and incorporated two of them into short peptide sequences.[45J,71 They demonstrated the formation of several metal complexes, in particular Rh complexes, and reported their structure as well as their ability to catalyze enantioselectively certain hydrogenation reactions.[481 While the enantioselectivities observed are modest so far, optimization through combinatorial synthesis will probably lead to useful catalysts. The synthesis of the sulfide protected form of both Fmoc- and Boc-dicyclohexylphosphinoserine 49 and -diphenylphosphinoserine 50 has been reported, in addition to diphenylphosphino-L-proline 51 (Scheme 14).[49 To show their compatibility with solid-phase peptide synthesis, they were incorporated into hydrophobic peptides, such as dodecapeptide 53, using the standard Fmoc protocol (Scheme 15).[451 For better results, the phosphine-modified amino acid 50 was coupled as a Fmoc-protected dipeptide 56, rather than the usual Fmoc derivative 52.[471 As an illustrative example, the synthesis of diphe-nylphosphinoserine 52 is depicted in Scheme 16J45 ... 

As in the case of Boc protection, the Fmoc group is not usually introduced on solid phase, but rather in solution, by the use of an activated Fmoc derivative (e.g. the chloroformate Fmoc-Cl or O-Fmoc-.V-hydroxysuccinimide, Fmoc-OSu) and aqueous base (Experimental Procedure 10.3)., V-/ lkylamino acids bound to cross-linked polystyrene have been Fmoc-protected by treatment with Fmoc-Cl (4 equiv.) and DIPEA (6 equiv.) in DCM for 2 h [132,259], Primary amines on insoluble supports can also be converted into Fmoc derivatives under these conditions [260]. 

Period 2 Part B—Work up the dansyl hydrolysate and spot on the TLC plate with standard dansyl amino acids. Part A. 1—Work up peptide hydrolysate and prepare FMOC derivatives of amino acids for analysis by HPLC or CE. Part A.2-If applicable, develop paper chromatogram in solvent system. 

Draw the structure of the FMOC derivative of the amino acid phenylalanine as it would exist at pH 7.0. 

S. Einarsson, B. Josefsson, and S. Lagerkvist,/. Chromatogr. 282, 609-618 (1983). FMOC derivatives of amino acids. 

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