Formation of Fmoc-beta-alanine during Fmoc-protections with Fmoc-OSu

During the Fmoc-protection of H-alpha-Me-Val-OH, an unknown side product was found and isolated. The characterization using various analytical methods led unambiguously to the result that Fmoc-beta-Ala-OH was formed during the reaction. The reagent Fmoc-OSu was proven to be the source of Fmoc-beta-Ala-OH, following a mechanism that involved many deprotonation and elimination steps and a Lossen-type rearrangement as key sequence. The impurity Fmoc-beta-Ala-OH was found in a variety of reactions in which Fmoc-OSu was applied, either in the reaction mixture or as a contamination of the crude product. Purification of the Fmoc-amino acid derivatives from this impurity incurred high costs and significant reductions in yield.     

Asparagine coupling in Fmoc solid phase peptide synthesis

To investigate side reactions during the activation of side chain unprotected asparagine in Fmoc-solid phase peptide synthesis the peptide Met-Lys-Asn-Val-Pro-Glu-Pro-Ser was synthesized using different coupling conditions for introduction of the asparagine residue. Asparagine was activated by DCC/HOBt, BOP (Castro's reagent) or introduced as the pentafluorophenyl ester. The resulting peptide products were analyzed by HPLC, mass spectrometry and Edman degradation. In the crude products varying amounts of beta-cyano alanine were found, which had been formed by dehydration of the side chain amide during carboxyl activation of Fmoc-asparagine. A homogeneous peptide was obtained by using either side chain protected asparagine derivatives with BOP mediated activation or by coupling of Fmoc-Asn-OPfp. Fmoc-Asn(Mbh)-OH and Fmoc-Asn(Tmob)-OH were coupled rapidly and without side reactions with BOP. For the side chain protected derivatives the coupling was as fast as that of other Fmoc-amino acid derivatives, whereas couplings of Fmoc-Asn-OH proceed more slowly. However, during acidolytic cleavage both protection groups, Mbh and Tmob, generate carbonium ions which readily alkylate tryptophan residues in a peptide. Tryptophan modification was examined using the model peptide Asn-Trp-Asn-Val-Pro-Glu-Pro-Ser. Alkylation could be reduced by addition of scavengers to the TFA during cleavage and side chain deprotection. A homogeneous peptide containing both, asparagine and tryptophan, was obtained only by coupling of Fmoc-Asn-OPfp.     

Solid-phase peptide synthesis using nanoparticulate amino acids in water.

Solid-phase peptide synthesis has many advantages compared with solution peptide synthesis. However, this procedure requires a large amount of organic solvents. Since safe organic solvent waste disposal is an important environmental problem, a technology based on coupling reaction of suspended nanoparticle reactants in water was studied. Fmoc-amino acids are used widely, but most of them show low solubility in water. We prepared well-dispersible Fmoc-amino acid nanoparticles in water by pulverization using a planetary ball mill in the presence of poly(ethylene glycol). Leu-enkephalin amide was prepared successfully using the nanoparticulate Fmoc-amino acid on a poly(ethylene glycol)-grafted Rink amide resin in water.     

Investigation of a failed DNA assay leads to identification of an unexpected contaminant in a commonly used chromatography buffer

For mammalian cell-derived recombinant biotherapeutics, controlling host cell DNA levels below a threshold is a regulatory requirement to ensure patient safety. DNA removal during drug substance manufacture is accomplished by a series of chromatography-based purification steps and a qPCR-based analytical method is most used to measure DNA content in the purified drug substance to enable material disposition. While the qPCR approach is mature and its application to DNA measurement is widespread in the industry, it is susceptible to trace levels of process-related contaminants that are carried forward. In this study, we observed failures in spike recovery studies that are an integral component of the qPCR-based DNA testing, suggesting the presence of an inhibitory compound in the sample matrix. We generated hypotheses around the origin of the inhibitory compound and generated multiple sample matrices and deployed a suite of analytical techniques including Raman and NMR spectroscopy to determine the origin and identity of the inhibitory compound. The caustic wash step and depth filter extractables were ruled out as root causes after extensive experimentation and DNA testing. Subsequently, 2-(N-morpholino)ethanesulfonic acid (MES), a buffer used in the chromatography unit operations, was identified as the source of the contaminant. A 500-fold concentration followed by Raman and NMR spectroscopy analysis revealed the identity of the inhibitory compound as polyvinyl sulfone (PVS), an impurity that originates in the MES manufacturing process. We have implemented PVS concentration controls for incoming MES raw material, and our work highlights the need for rigor in raw material qualification and control.     

Synthetic strategy for side chain mono-N-alkylation of Fmoc-amino acids promoted by molecular sieves

A new synthetic strategy to alkylate amino groups under mild conditions has been developed. It utilizes only 4 ? molecular sieves as base in order to promote the N-alkylation reaction, in presence of the appropriate alkyl halide. The methodology was validated by the simple and efficient side-chain N-alkylation of o-Ns-protected Fmoc-amino acid. One of them was introduced as building block into a peptide sequence, thus allowing the preparation of site-specific alkylated peptide molecules.     

Tracing and control of raw materials sourcing for vaccine manufacturers

The control of the raw materials used to manufacture vaccines is mandatory; therefore, a very clear process must be in place to guarantee that raw materials are traced. Those who make products or supplies used in vaccine manufacture (suppliers of culture media, diagnostic tests, etc.) must apply quality systems proving that they adhere to certain standards. ISO certification, Good Manufacturing Practices for production sites and the registration of culture media with a ‘Certificate of Suitability’ from the European Directorate for the Quality of Medicines and Healthcare are reliable quality systems pertaining to vaccine production. Suppliers must assure that each lot of raw materials used in a product that will be used in vaccine manufacture adheres to the level of safety and traceability required. Incoming materials must be controlled in a single ‘Enterprise Resource Planning’ system which is used to document important information, such as the assignment of lot number, expiration date, etc. Ingredients for culture media in particular must conform to certain specifications. The specifications that need to be checked vary according to the ingredient, based on the level of risk.The way a raw material is produced is also important, and any aspect relative to cross-contamination, such as the sanitary measures used in producing and storing the raw material must be checked as well. In addition, suppliers can reduce the risk of viral contamination of raw materials by avoiding purchases in countries where a relevant outbreak is currently declared.     

The oxidative damage of plasmid DNA by ascorbic acid derivatives in vitro: the first research on the relationship between the structure of ascorbic acid and the oxidative damage of plasmid DNA

To study the structure-function relationship of the oxidative-damage effect of ascorbic acid, we have focused on the interaction between plasmid DNA pUC19 and a series of ascorbic acid derivatives modified on different OH groups in the presence of transition metal ions. Some ascorbic acid derivatives can selectively cleave plasmid DNA from Form I to Form II in the presence of low concentration of Cu2+ just like ascorbic acid itself, while other derivatives oxidatively damage plasmid DNA slightly. We found that those derivatives with unattached 2-OH and 3-OH groups retain the ability to cleave the plasmid DNA. The derivatives that have been methylated on 2-OH or 3-OH can only cleave plasmid DNA softly, and those derivatives that have been protected on both 2-OH and 3-OH can hardly exert an oxidative damage on plasmid DNA under the same condition. Form these results, we can draw the conclusion that 2-OH and 3-OH groups of the ascorbic acid molecule contribute most to this biological activity.     

Synthesis and application of acid labile anchor groups for the synthesis of peptide amides by Fmoc-solid-phase peptide synthesis

The preparation and application of a new linker for the synthesis of peptide amides using a modified Fmoc-method is described. The new anchor group was developed based on our experience with 4,4'-dimethoxybenzhydryl (Mbh)-protecting group for amides. Lability towards acid treatment was increased dramatically and results in an easy cleavage procedure for the preparation of peptide amides. The synthesis of N-9-fluorenylmethoxycarbonyl- ([5-carboxylatoethyl-2.4-dimethoxyphenyl)- 4'-methoxyphenyl]-methylamin is reported in detail. This linker was coupled to a commercially available aminomethyl polystyrene resin. Peptide synthesis proceeded smoothly using HOOBt esters of Fmoc-amino acids. Release of the peptide amide and final cleavage of the side chain protecting groups was accomplished by treatment with trifluoroacetic acid-dichloromethane mixtures in the presence of scavengers. The synthesis of peptide amides such as LHRH and C-terminal hexapeptide of secretin are given as examples.     

Application of 2-chlorotrityl resin in solid phase synthesis of (Leu15)-gastrin I and unsulfated cholecystokinin octapeptide. Selective O-deprotection of tyrosine.

The carboxyl terminal dipeptide amide, Fmoc-Asp-Phe-NH2, of gastrin and cholecystokinin (CCK) has been attached in high yield through its free side chain carboxyl group to the acid labile 2-chlorotrityl resin. The obtained peptide resin ester has been applied in the solid phase synthesis of partially protected (Leu15)-gastrin I utilising Fmoc-amino acids. Quantitative cleavage of this peptide from resin, with the t-butyl type side chain protection intact is achieved using mixtures of acetic acid/trifluoroethanol/dichloromethane. Under the same conditions complete detritylation of the tyrosine phenoxy function occurs simultaneously. Thus, the solid-phase synthesis of peptides selectively deprotected at the side chain of tyrosine is rendered possible by the use of 2-chlorotrityl resin and Fmoc-Tyr(Trt)-OH. The efficiency of this approach has been proved by the subsequent high-yield synthesis of three model peptides and the CCK-octapeptide.     

Reduction of a 4-pyrrole phenylacyl-containing peptide with trifluoroacetic acid-triisopropylsilane-phenol-H2O during solid-phase peptide synthesis and its protein kinase C alpha inhibitory activity

Cleavage of 4-pyrrole phenylacyl TentaGel-appended peptide (5) containing Arg(Pbf) with trifluoroacetic acid/triisopropylsilane/phenol/H2O (90/2/4/4) gives the 4-pyrrole phenylacyl peptide (3). However, cleavage of 4-pyrrole phenylacyl Rink-appended peptide (7) containing Arg(Pbf) using the same reagents furnishes the 4-pyrrolidine phenylacyl peptide (8), which contains the reduced pyrrole ring. Compound 8 displays a Ki of 2.32 microM, approximately fivefold less potent than compound 3.     

Removal of acid-labile protecting or anchoring groups in the presence of polyfluorinated alcohol: Application to solid-phase peptide synthesis

We describe herein a new method for cleaving from resin and removing acid-labile protecting groups in solid-phase peptide synthesis in the presence of a polyfluorinated alcohol (either trifluoroethanol, TFE, or hexafluoroisopropanol, HFIP). It was shown that 0.1 M HCl in hexafluoroisopropanol or trifluoroethanol removes the acid-labile protecting groups commonly used in Fmoc SPPS for the protection of amino acid side-chains, such as t-butyl ester and ether, Boc, trityl, and Pbf groups including the most acid-resistant p-hydroxymethylphenoxyacetyl group (HMPA), p-benzyloxy benzyl ester (Wang resin), Rink amide, and peptide amide linker (PAL). The addition of 5–10% of a hydrogen-bonding solvent was shown to considerably retard or even fully inhibit the reaction. However, nonhydrogen-bonding solvents, such as dichloromethane, do not slow down the reaction.     

Facile solid-phase synthesis of C-terminal peptide aldehydes and hydroxamates from a common Backbone Amide-Linked (BAL) intermediate.

C-Terminal peptide aldehydes and hydroxamates comprise two separate classes of effective inhibitors of a number of serine, aspartate, cysteine, and metalloproteases. Presented here is a method for preparation of both classes of peptide derivatives from the same resin-bound Weinreb amide precursor. Thus, 5-[(2 or 4)-formyl-3,5-dimethoxyphenoxy]butyramido-polyethylene glycol-polystyrene (BAL-PEG-PS) was treated with methoxylamine hydrochloride in the presence of sodium cyanoborohydride to provide a resin-bound methoxylamine, which was efficiently acylated by different Fmoc-amino acids upon bromo-tris-pyrrolidone-phosphonium hexafluorophosphate (PyBrOP) activation. Solid-phase chain elongation gave backbone amide-linked (BAL) peptide Weinreb amides, which were cleaved either by trifluoroacetic acid (TFA) in the presence of scavengers to provide the corresponding peptide hydroxamates, or by lithium aluminum hydride in tetrahydrofuran (THF) to provide the corresponding C-terminal peptide aldehydes. With several model sequences, peptide hydroxamates were obtained in crude yields of 68-83% and initial purities of at least 85%, whereas peptide aldehydes were obtained in crude yields of 16-53% and initial purities in the range of 30-40%. Under the LiAlH4 cleavage conditions used, those model peptides containing t-Bu-protected aspartate residues underwent partial side chain reduction to the corresponding homoserine-containing peptides. Similar results were obtained when working with high-load aminomethyl-polystyrene, suggesting that this chemistry will be generally applicable to a range of supporting materials.     

Preparation and application of O-amino-serine, Ams, a new building block in chemoselective ligation chemistry.

The non-codable amino acid O-amino-serine, Ams, has been prepared in both L- and D-forms as the orthogonally protected derivative, Fmoc-Ams(Boc)-OH (1 and 2). This new amino acid derivative is useful for chemoselective ligations. Under acidic conditions and in the presence of all other common amino acid functionalities, the oxyamine function selectively forms oxime linkages with aldehydes. The Ams residue has been incorporated into both ends of the peptide sequence Asp-Leu-Trp-Gln-Lys using standard SPPS. The deprotected peptide has been used for chemical ligation to afford a peptide dimer as well as a glycopeptide. Ams racemization was found to be negligible, as monitored by HPLC separation of Ams dipeptide diastereomers.     

Synthesis of Fmoc-amino acid chlorides assisted by ultrasonication, a rapid approach

The chloride derivatives of all common Fmoc-amino acids lacking polar side chains as well as a number of benzyl based side chain protection have been prepared using the corresponding amino acid and thionyl chloride assisted by ultrasound has been described. The protocol is simple, efficient and rapid. All the acid chlorides prepared have been obtained in good yield and purity.     

In vivo effects of gamma-aminobutyric acid and beta-alanine on thyrotrophin secretion in the normal and hypothyroid rat

The effect of pharmacological doses of two amino acids neurotransmitters, gamma-aminobutyric acid (GABA) and beta-alanine (beta-Ala), on thyrotrophin (TSH) secretion was studied in normal and hypothyroid (PTU-treated) male rats. Inhibition of TSH secretion was observed in normal rats treated with the drugs, 30 min after their administration. Hypothyroid animals responded only to GABA administration, decreasing their serum TSH at 30 min. Response to thyrotrophin-releasing hormone (TRH) after 15 min of drug administration was blunted in GABA injected animals, as compared to saline-injected controls. When TRH was injected at the same time as GABA and beta-Ala, the response was significantly lower than in controls. It is suggested that beta-Ala and GABA act at the pituitary by impairing the TSH response to TRH. The possibility that beta-Ala actions may be due to decreased GABA catabolism is considered, since beta-Ala administration increased GABA synaptosomal levels.     

Synthesis of Fmoc-amino acid chlorides assisted by ultrasonication,a rapid approach

Summary The chloride derivatives of all common Fmoc-amino acids lacking polar side chains as well as a number of benzyl based side chain protection have been prepared using the corresponding amino acid and thionyl chloride assisted by ultrasound has been described. The protocol is simple, efficient and rapid. All the acid chlorides prepared have been obtained in good yield and purity.     

Further studies on the use of 2,2,2-trichloroethyl groups for phosphate protection in phosphoserine peptide synthesis.

Boc-Ser(PO3Tc2)-OH, Z-Ser(PO3Tc2)-OH and Fmoc-Ser(PO3Tc2)-OH, derivatives useful for peptide synthesis, have been obtained in high yields by acylation of H-Ser(PO3Tc2)-OH.CF3COOH. The latter was obtained from Boc- or Z-Ser(PO3Tc2)-OBzl by simultaneous removal of the amino- and carboxy-protecting groups by Pd-catalyzed hydrogenolysis in acetic acid-trifluoroacetic acid solution. Removal of the Tc-protecting group was efficiently achieved by hydrogenolysis in aqueous ethanol.     

Proteinogenic amino acids labelled with 15N and/or 13C for application in peptide synthesis: A short review with a comprehensive list of published derivatives

A review is given of the literature dealing with the most common protected derivatives of ¹⁵N- and/or ¹³C-labelled amino acids of interest in peptide synthesis. The list contains all such Boc-, Z- and Fmoc-amino acids as well as published methyl, ethyl, t-butyl and benzyl esters.     

Entrapping unusual folding characteristics of the beta-Ala residues in a model peptide: Boc-beta-Ala-Aib-beta-Ala-NHCH3.

Crystal structure analysis of a model peptide: Boc-beta-Ala-Aib-beta-Ala-NHCH3 (beta-Ala: 3-amino propionic acid; Aib: alpha-aminoisobutyric acid) revealed distinct conformational preferences for folded [phi approximately 136 degrees, mu approximately -62 degrees, psi approximately 100 degrees] and semifolded [phi approximately 83 degrees, mu approximately -177 degrees, psi approximately -117 degrees] structures of the N-and C-terminus beta-Ala residues, respectively. The overall folded conformation is stabilized by unusual Ni...H-Ni+1 and nonconventional C-H...O intramolecular hydrogen bonding interactions.     

Synthesis of O-glycosylated tuftsins by utilizing threonine derivatives containing an unprotected monosaccharide moiety

Synthesis is described of four tuftsin derivatives containing a D-glucopyranosyl or a D-galactopyranosyl unit covalently linked to the hydroxy side chain function of the threonine residue through either an alpha or beta O-glycosidic linkage. Fmoc-threonine derivatives containing the suitable unprotected sugar were used for incorporating the O-glycosylated amino acid residue. Z-Thr[alpha-Glc(OBzl)4]-OBzl and Z-Thr[alpha-Gal(OBzl)4]-OBzl were prepared from the tetra-O-benzylated sugar and Z-Thr-OBzl by the trichloroacetimidate method in the presence of trimethylsilyl trifluoromethane sulfonate. The alpha glycosylated threonine derivatives were converted into Fmoc-Thr(alpha-Glc)-OH and Fmoc-Thr(alpha-Gal)-OH by catalytic hydrogenation followed by acylation with Fmoc-OSu. beta-Glucosylation and beta-galactosylation of threonine were carried out by reacting the proper per-O-acetylated sugar with Z-Thr-OBzl and boron trifluoride ethyl etherate in dichloromethane. Catalytic hydrogenation of the beta-O-glycosylated threonine derivatives followed by acylation with Fmoc-OSu and deacetylation with methanolic hydrazine yielded Fmoc-Thr(beta-Glc)-OH and Fmoc-Thr(beta-Gal)-OH, respectively. The O-glycosylated threonine derivatives were then reacted with H-Lys(Z)-Pro-Arg(NO2)-OBzl in the presence of DCC and HOBt and the resulting glycosylated tuftsin derivatives were fully deblocked by catalytic hydrogenation, purified by HPLC, and characterized by optical rotation, amino acid analysis, and 1H NMR. The beta-galactosylated tuftsin was also prepared by the continuous flow solid phase procedure.