Peptide permethylated

Alkylation of phenols, alcohols, amides, and acids. N-Alkylation of indoles and pyrroles by means of solid KOH in DMSO was reported a few years ago. Actually this method is applicable to a number of substrates. The substrate and alkyl halide are added to powdered KOH and stirred in DMSO, usually at 20°. Methyl-ation of phenols, alcohols, and amides occurs in high yield in about 5-30 minutes. Esterification of acids is slower. Dehydrohalogenation is a competing or predominating reaction when secondary or tertiary halides are used. Another limitation is that amino groups are converted into quaternary salts under these conditions. The general method can be used for permethylation of peptides. 

Several factors affect the volatility and stability of a peptide derivative, not least of these being the number and nature of the constituent amino acids. Heterocyclic and aromatic amino acids reduce volatility while those containing sulphur tend to decrease the thermal stability. Small naturally occurring peptides which are not derived from proteins often contain only aliphatic amino acids which lack functional groups in the side chains. Peptides of this type of up to about ten amino acids, after conversion to suitable derivatives, are amenable to analysis by mass spectrometry, e.g. [164]. A variety of derivatives has been reported and include N-trifluoroacetyl peptide esters [136,165], N-acetyl peptide esters [166-168], aromatic N-acyl peptide esters [169-172], and per-methylated N-acyl peptides [173]. The principal modes of the electron impact induced fragmentation of these peptide derivatives are well established and have been summarised in recent reviews [174,175]. Although the spectra of the permethylated derivatives [176] are perhaps the simplest and easiest to interpret and are now frequently used, the N-acyl peptide esters have been widely and successfully employed. 

Figure 1.13 shows the spectrum of a hexapeptide [181] as its permethy-lated derivative with the ions indicating the amino acid order. The rapid decrease in the intensity of the important sequence-determining ions at higher mass is evident and a principal reason why MS often requires several times as much material as the common micro wet chemical methods for sequencing small peptides. The chemical ionization mass spectra of some N -acyl permethylated simple peptides show a much more even distribution of the sequencing peaks and hence require a lower sample level than for El spectra [187]. This may well prove of value in the future. Cl has also been applied directly to peptides [188] where up to six amino acids units have been introduced by the direct insertion probe. 

Gray [192] proposed a method in which by acetylating the initial protein the terminal peptide liberated by specific enzymic degradation was isolated and its permethylated derivative sequenced by mass spectrometry. 

The analysis of peptides and proteins has played a role in the development of LC-MS from the early 1980s. Yu et al. [1] reported the analysis of a Y-acetyl-Y,0,5 -permethylated octapeptide derived from the C-terminus of glucagon as early as 1984, using a moving-belt interface and isobutane chemical ionization. Stroh et al. [2] applied FAB and the moving-belt interface to the stracture elucidation of antibiotic leucinostatin peptides and a tryptic digest of antiamoebin 1. 

At the beginning, permethylated peptide libraries were derived by a post-modification of peptide libraries attached to the solid-phase support. The amide bond functionalities were alkylated using a solution of sodium hydride in dimethylsulfoxide, followed by addition of neat methyl iodide, therefore generating libraries from libraries . A peptide library that underwent this simple chemical transformation was used to identify compounds with potent antimicrobial activity against Gram-positive bacteria [21]. 

In 1967 E. Lederer (Plate 28) and B.C. Das reported [32] that the volatility of acetyl-peptide esters can be increased by replacing all dissociable protons with methyl groups. This permethylation can be performed with methyl iodide in anhydrous dimethylsulfoxide in the presence of a strong base. In iV-methyl peptides the coherence caused by hydrogen bonds is overcome. Moreover, the fragmentation patterns are much simpler than the patterns found in N-acetyl peptide esters without permethylation. Fragmentation occurs almost exclusively at the peptide bonds and the mass spectra consist mainly of sequence peaks of high intensity (Fig. 14). 

P. Roepstorff, K. Norris, S, Severinsen, K. Brunfeldt, Mass spectrometry of peptide derivatives. Temporary protection of methionine as sulfoxide during permethylation FEBS Lett. 9 235-238(1970)... 

Scheme 1. Chemical ionization of a permethylated peptide and fragmentation of the peptide bond...

Das and Nebelin (11, 52a) have checked the structures of several MDP analogues permethylated peptides (10, 68) were analyzed by electron impact mass spectrometry. 

Fig. 11 shows the structure of permethylated MDP with the principal fragmentations (dotted lines) Fig. 12 shows the corresponding mass spectrum. The molecular ion peak is at m/e 618 and a sequence peak at m/e 417, accompanied by m/e 389 (loss of CO) and 357 (loss of MeOH). Loss of the entire pyranose ring gives the ion at m/e 342. The complementary C-terminal fragment to m/e 417 is at m/e 203. Peaks at m/e 260 and 228 (260-MeOH) contain the intact pyranose ring and are known to occur in the spectra of permethylated-N-acetyl hexosamines. In these fragments the entire lactyl peptide chain has been eliminated. 

Many matrices have been used for carbohydrate analysis but 2,5-DHB has proved to be the most versatile and widely used. Esculetin is more specific, however, and ionizes sugars in preference to peptides and glycopeptides in mixtures of these compounds (Figure 4). Carbohydrates form [M -I- Na] ions with most matrices but other adducts can be produced if the matrix is doped with the appropriate salt. Anionic carbohydrates additionally form [M — H] ions. Sensitivity in both modes is lower than with peptides as the latter can be efficiently protonated because of their high proton affinity. However, sensitivity can be increased by permethylation or derivatization at the reducing terminus with a compound that can be protonated, such as a tertiary amine or one that possesses a constitutive charge. Alkyltrimethylammonium compounds have been used in the latter context. 

Peak B is closely related to a-MSH it has an identical amino acid composition and electron impact mass spectrum (AT-acetyl-Ar,0,S-permethyl derivative) and is converted into a-MSH on base hydrolysis (Fig. 9). Using the nascent mass spectrometric technique of fast atom bombardment (FAB) (Barber et al, 1981 Morris et al., 1981), the molecular weight of peak B was shown to be 42 mass units higher than a-MSH. These data together with EIMS analysis of underivatized peptides obtained by proteolytic digestion of peak B, allowed us to determine its structure as bis acetyl (Ser 1) a-MSH. 

Alkylation of Amides, Phenols, Alcohols, and Adds. A variety of carboxamides were alkylated with primary alkyl halides using KOH in DMSO to give the fYalkyl amides (eq 1) in 54—90% yield. Most reactions were carried out at rt, but in some cases heating to 90 °C was required. Similar conditions were applied to alcohols, phenols, and acids to form ethers and esters. The procedure applies to Mel and all primary halides. Secondary alkyl halides show evidence of competitive dehydrohalogenation, while tertiary halides do not give any alkylation products. The procedure was applied to the N- and O-permethylation of peptides. It was also applied to the methylation of hydroxypyridines in 39-78% yield. In all the above cases, the substrate and alkyl halide were added to powdered KOH in DMSO and stirred at rt. It was unnecessary to use especially dry DMSO or to protect the reaction mixture from atmospheric moisture. 

Partial acid hydrolysis gave an amide of the P-amino fatty acid and serine, with an N-terminal position for the serine residue. The structures of several other peptides were established by chemical means and by mass spectrometry of their permethylated derivatives. From these results, the complete structure of iturin A was established as (57) (138). Circular dichroism of derivatives of the P-amino fatty acids (iturinic acids) showed their configurations to be R 139). 

---