Identification of Fmoc‐β‐Ala‐OH and Fmoc‐β‐Ala‐amino acid‐OH as new impurities in Fmoc‐protected amino acid derivatives*

Abstract: During the manufacture of a proprietary peptide drug substance a new impurity appeared unexpectedly. Investigation of its chemical structure established the impurity as a β‐Ala insertion mutant of the mother peptide. The source of the β‐Ala was identified as contamination of the Fmoc‐Ala‐OH raw material with Fmoc‐β‐Ala‐Ala‐OH. Further studies also demonstrated the presence of β‐Ala in other Fmoc‐amino acids, particularly in Fmoc‐Arg(Pbf)‐OH. In this case, it was due to the presence of both Fmoc‐β‐Ala‐OH and Fmoc‐β‐Ala‐Arg(Pbf)‐OH. It is concluded that β‐Ala contamination of Fmoc‐amino acid derivatives is a general and hitherto unrecognized problem to suppliers of Fmoc‐amino acid derivatives. The β‐Ala is often present as Fmoc‐β‐Ala‐OH and/or as a dipeptide, Fmoc‐β‐Ala‐amino acid‐OH. In collaboration with the suppliers, new specifications were introduced, recognizing the presence of β‐Ala‐related impurities in the raw materials and limiting them to acceptable levels. The implementation of these measures has essentially eliminated β‐Ala contamination as a problem in the manufacture of the drug substance.     

An efficient and highly selective deprotection of N‐Fmoc‐α‐amino acid and lipophilic N‐Fmoc‐dipeptide methyl esters with aluminium trichloride and N,N‐dimethylaniline

Abstract: A novel procedure for the deprotection of the carboxyl group of amino acid methyl esters is presented. The process is carried out by the reagent system aluminium trichloride/N,N‐dimethylaniline that can successfully be applied to unblock the carboxyl moiety either of N‐Fmoc‐protected amino acid methyl esters and N‐Fmoc‐protected short dipeptide methyl esters. The chiralities of the optically pure amino acid or peptide precursors are maintained totally unchanged.     

Convenient and efficient synthesis of Boc‐/Z‐/Fmoc‐β‐amino acids employingN‐protected α‐amino acid fluorides

Abstract: A new and efficient method for the synthesis ofN^α‐Fmoc‐/Boc‐/Z‐β‐amino acids using the two‐step Arndt‐Eistert approach is described. Fmoc‐/Boc‐/Z‐α‐Amino acid fluorides were used for the acylation of diazomethane synthesizing Fmoc‐/Boc‐/Z‐α‐aminodiazoketones as crystalline solids with good yield and purity. They were then converted to the corresponding β‐amino acids using PhCOOAg/dioxane/H₂O.     

Facile synthesis of orthogonally protected amino acid building blocks for combinatorial N‐backbone cyclic peptide chemistry

Abstract: Protected N^α‐(aminoallyloxycarbonyl) and N^α‐(carboxyallyl) derivatives of all natural amino acids (except proline), and their chiral inverters, were synthesized using facile and efficient methods and were then used in the synthesis of N^α‐backbone cyclic peptides. Synthetic pathways for the preparation of the amino acid building units included alkylation, reductive amination and Michael addition using alkylhalides, aldehydes and α,β‐unsaturated carbonyl compounds, and the corresponding amino acids. The resulting amino acid prounits were then subjected to Fmoc protection affording optically pure amino acid building units. The appropriate synthetic pathway for each amino acid was chosen according to the nature of the side‐chain, resulting in fully orthogonal trifunctional building units for the solid‐phase peptide synthesis of small cyclic analogs of peptide loops (SCAPLs^?). N^α‐amino groups of building units were protected by Fmoc, functional side‐chains were protected by t‐Bu/Boc/Trt and N‐alkylamino or N‐alkylcarboxyl were protected by Alloc or Allyl, respectively. This facile method allows easy production of a large variety of amino acid building units in a short time, and is successfully employed in combinatorial chemistry as well as in large‐scale solid‐phase peptide synthesis. These building units have significant advantage in the synthesis of peptido‐related drugs.     

Evaluation of new base‐labile2‐(4‐nitrophenylsulfonyl)ethoxycarbonyl (Nsc)‐amino acids for solid‐phase peptide synthesis

Abstract: The 2‐(4‐nitrophenylsulfonyl)ethoxycarbonyl (Nsc) group is a new base‐labile protecting group for solid‐phase peptide synthesis, completely interchangeable with the fluorenylmethoxycarbonyl (Fmoc) protecting group, but with certain advantages. In this paper, we report a methodology with N^α‐Nsc‐protected amino acids for the synthesis of some melanotropins important to our research, namely, γ‐melanocyte‐stimulating hormone (γ‐MSH), its [Nle³]‐analogue, and a cyclic α‐MSH/β‐MSH hybrid. We developed an efficient protocol for the synthesis of the cyclic MSH analogue that yielded this peptide in > 98% purity. The γ‐MSH synthesis, which gave problems with both the Boc and Fmoc strategies, yielded the desired peptide by Nsc‐chemistry but was accompanied by side products. Finally, the Nle³‐γ‐MSH analogue was synthesized more efficiently using the Fmoc strategy, suggesting that Nsc‐chemistry might not be the best methodology for certain sequences.     

Simple and stereospecific homologation of urethane‐protected α‐amino acids to their higher homologs using HBTU1

Abstract: A simple, efficient and stereospecific approach for the homologation of urethane‐protected α‐amino acids to β‐amino acids by the Arndt–Eistert method employing Fmoc‐/Boc‐α‐amino acid and 2‐(1H‐benzotriazole‐1‐yl)‐1,1,3,3‐tetramethyl‐uronium hexafluorophosphate mixture for the acylation of diazomethane synthesizing the key intermediates Fmoc‐/Boc‐α‐aminodiazomethanes as crystalline solids is described.     

A solid‐phase synthetic strategy for the preparation of peptide‐based affinity labels: synthesis of dynorphin A analogs

Abstract: Solid‐phase synthetic methodology was developed for the preparation of peptide‐based affinity labels. The initial peptides synthesized were dynorphin A (Dyn A) analogs [Phe(p‐X)⁴,d ‐Pro¹⁰]Dyn A(1–11)NH₂ containing isothiocyanate (X = –N=C=S) and bromoacetamide (X = –NHCOCH₂Br) groups. The peptides were assembled on solid supports using Fmoc‐protected amino acids, and the side chain amine to be functionalized, Phe(p‐NH₂), was protected by the Alloc (allyloxycarbonyl) group. Following removal of the Alloc group by palladium(0), the reactive isothiocyanate and bromoacetamide functionalities were successfully introduced while the peptides were still attached to the resin. Synthesis of these peptides was carried out on polystyrene (PS) and polyethylene glycol–polystyrene (PEG–PS) resins containing the PAL [peptide amide linker, 5‐(4‐Fmoc‐aminomethyl‐3,5‐dimethoxyphenoxy)valeric acid] linker. Both the rate of Alloc deprotection and the purity of the crude affinity‐labeled peptides obtained were found to be dependent on the resin used for peptide assembly.     

Nsc and Fmoc Nα‐amino protection for solid‐phase peptide synthesis: a parallel study

Abstract The 2‐(4‐nitrophenylsulfonyl)ethoxycarbonyl (Nsc) group is an alternative to Fmoc for N^α‐protection in solid‐phase peptide synthesis. Nsc‐amino acids may be particularly suitable for automatic synthesizers, in which the amino acids are stored in solution, and the incorporation of residues prone to racemization such as Cys and His. Owing to the hydrophilicity of the Nsc group, these derivatives are useful for the preparation of protected peptides in convergent solid‐phase peptide synthesis strategies.     

Fmoc/Trt-amino acids: comparison to Fmoc/tBu-amino acids in peptide synthesis

Model peptides containing the nucleophilic amino acids Trp and Met have been synthesized with the application of Fmoc/Trt- and Fmoc/tBu-amino acids, for comparison. The deprotection of the peptides synthesized using Fmoc/Trt-amino acids in all cases leads to crude peptides of higher purity than that of the same peptides synthesized using Fmoc/tBu-amino acids. © Munksgaard 1998.     

Combined solid‐phase/solution synthesis of large ribonuclease A C‐terminal peptides containing a non‐natural proline analog*

Abstract: Three large peptides corresponding to the 65–124 (60‐mer), 72–124 (53‐mer), and 77–124 (48‐mer) sequence of bovine pancreatic ribonuclease A (RNase A) were assembled from either two or three shorter protected peptide fragments by chemical coupling in solution. The fragments were synthesized manually by 9‐fluorenylmethyloxycarbonyl (Fmoc)‐based solid‐phase peptide chemistry in plastic syringes, and subsequently purified by normal‐phase high‐performance liquid chromatography on a silica gel column. The main aim of this work was to incorporate sterically hindered l ‐5,5‐dimethylproline (dmP) as a substitute for Pro⁹³ into the sequence of RNase A in order to constrain the –Tyr⁹²‐Pro⁹³– peptide group to a single cis‐conformation.     

Synthesis of novel protected Nα(ω‐thioalkyl) amino acid building units and their incorporation in backbone cyclic disulfide and thioetheric bridged peptides

Abstract: General methods for the preparation of protected N^α(ω‐thioalkyl) amino acids building units for backbone cyclization using reductive alkylation and on‐resin preparation are described. The synthesis of non‐Gly Fmoc‐protected S‐functionalized N‐alkylated amino acids is based on the reaction of readily prepared protected ω‐thio aldehyde with the appropriate amino acid. Preparation of Fmoc‐protected S‐functionalized N‐alkylated Gly building units was carried out using two methods: reaction of glyoxylic acid with Acm‐thioalkylamine and an on‐resin reaction of bromoacetyl resin with Trt‐thioalkylamines. Three model peptides were prepared using these building units. The GlyS2 building unit was incorporated into a backbone cyclic analog of somatostatin that contains a disulfide bridge. Formation of the disulfide bridge was performed by on‐resin oxidation using I₂ or Tl(CF₃COO^–)₃. Both methods resulted in the desired product in a high degree of purity in the crude. The AspS3 building unit was also successfully incorporated into a model peptide. In addition, the in situ generation of sulfur containing Gly building units was demonstrated on a Substance P backbone cyclic analog containing a thioether bridge.     

Synthesis and biodistribution studies of iodine‐131 D‐amino acid YYK peptide as a potential therapeutic agent for labeling an anti‐CD20 antibody

A major drawback of conventionally radioiodinated monoclonal antibodies for radioimmunotherapy is in vivo dehalogenation of iodine as a result of deiodinase recognition. To solve this problem we have synthesized a YYK tri‐peptide consisting of non‐metabolizable D ‐amino acids modified with the N‐succinimidyl (N‐Succ) function. The chemical purity of the synthesized peptide as assessed by analytical high performance liquid chromatography was 95%. Labeling of the Fmoc‐D ‐Tyr(_tBu)‐D ‐Tyr(_tBu)‐D ‐Lys(Boc)‐N‐Succ was performed using the chloramine‐T method and the conventional extraction, resulting in a radiochemical yield of 50–71% and a radiochemical purity of >95%. Radioiodination of the peptide was followed by conjugation to anti‐CD20 antibody with 65–75% labeling efficiency and 90% radiochemical purity. The effect of radioiodinated peptide on the biological behavior of the conjugate was evaluated through biodistribution studies in normal Lewis rats. Thyroid and stomach levels from Rituximab labeled with [¹³¹I]‐YYK‐peptide were two‐ to four‐fold less than those with directly labeled [¹³¹I]‐Rituximab, suggesting low recognition of its D ‐iodotyrosine residue by endogenous deiodinases. The favorable in vitro/in vivo stability and biodistribution profiles suggest that this radioiodine‐labeled YYK peptide is a good candidate for further exploration of its potential clinical application. Copyright © 2009 John Wiley & Sons, Ltd.     

Solid‐phase synthesis of peptides containing the spin‐labeled 2,2,6,6‐tetramethylpiperidine‐1‐oxyl‐4‐amino‐4‐carboxylic acid (TOAC)

Abstract: 2,2,6,6‐Tetramethylpiperidine‐1‐oxyl‐4‐amino‐4‐carboxylic acid (TOAC) is a nitroxide spin‐labeled, achiral C^α‐tetrasubstituted amino acid recently shown to be not only an effective β‐turn and 3₁₀/α‐helix promoter in peptides, but also an excellent rigid electron paramagnetic resonance probe and fluorescence quencher. Here, we demonstrate that TOAC can be effectively incorporated into internal positions of peptide sequences using Fmoc chemistry and solid‐phase synthesis in an automated apparatus.     

Synthesis and evaluation of potential affinity labels derived from endomorphin‐2

Abstract: In an attempt to identify potential peptide‐based affinity labels for opioid receptors, endomorphin‐2 (Tyr‐Pro‐Phe‐PheNH₂), a potent and selective endogenous ligand for µ‐opioid receptors, was chosen as the parent peptide for modification. The tetrapeptide analogs were prepared using standard Fmoc‐solid phase peptide synthesis in conjunction with incorporation of Fmoc‐Phe(p‐NHAlloc) and modification of the p‐amino group. The electrophilic groups isothiocyanate and bromoacetamide were introduced into the para position on either Phe³ or Phe⁴; the corresponding free amine‐containing peptides were also prepared for comparison. The peptides bearing an affinity label group and their free amine analogs were evaluated in a radioligand‐binding assay using Chinese hamster ovary (CHO) cells expressing µ‐ and δ‐opioid receptors. Modification on Phe⁴ was better tolerated than on Phe³ for µ‐receptor binding. Among the analogs tested, [Phe(p‐NH₂)⁴]endomorphin‐2 showed the highest affinity (IC₅₀ = 37 nm ) for µ‐receptors. The Phe(p‐NHCOCH₂Br)⁴ analog displayed the highest µ‐receptor affinity (IC₅₀ = 158 nm ) among the peptides containing an affinity label group. Most of the compounds exhibited negligible binding affinity for δ‐receptors, similar to the parent peptide.     

Preparation of oligomer-free Nα-Fmoc and Nα-urethane amino acids

A variety of N^α-urethane blocked amino acids, in particular 9-fluorenylmethyloxycarbonyl (Fmoc) derivatives, have been synthesized by utilizing intermediate O,N-bis-trimethylsilyl-amino acids, formed in situ by treating an amino acid with trimethylsilylchloride and a base in an aprotic solvent. The intermediate is then reacted with an acylating agent. A general procedure is given which eliminates the oligomerization side reactions normally observed in Schotten-Baumann type methods. Protected amino acids obtained from this procedure are of high purity as judged by t.l.c, HPLC, ion exchange chromatography, and other physical parameters.     

Improved synthesis of 5‐hydroxylysine (Hyl) derivatives*

Abstract: The synthesis of 5‐hydroxylysine (Hyl) derivatives for incorporation by solid‐phase methodologies presents numerous challenges. Hyl readily undergoes intramolecular lactone formation, and protected intermediates often have poor solubilities. The goals of this work were twofold: first, develop a convenient method for the synthesis of O‐protected Fmoc‐Hyl; secondly, evaluate the efficiency of methods for the synthesis of O‐glycosylated Fmoc‐Hyl. The 5‐O‐tert‐butyldimethylsilyl (TBDMS) fluoren‐9‐ylmethoxycarbonyl‐Hyl (Fmoc‐Hyl) derivative was conveniently prepared by the addition of tert‐butyldimethylsilyl trifluoromethanesulfonate to copper‐complexed Hyl[?‐tert‐butyloxycarbonyl (Boc)]. The complex was decomposed with Na⁺ Chelex resin and the Fmoc group added to the α‐amino group. Fmoc‐Hyl(?‐Boc, O‐TBDMS) was obtained in 67% overall yield and successfully used for the solid‐phase syntheses of 3 Hyl‐containing peptides. The preparation of Fmoc‐Hyl[?‐Boc, O‐(2,3,4,6‐tetra‐O‐acetyl‐β‐d ‐galactopyranosyl)] was compared for the thioglycoside, trichloroacetimidate and Koenigs–Knorr methods. The most efficient approach was found to be Koenigs–Knorr under inverse conditions, where Fmoc‐Hyl(?‐Boc)‐OBzl and peracetylated galactosyl bromide were added to silver trifluoromethanesulfonate in 1,2‐dichloroethane, resulting in a 45% isolated yield. Side‐reactions that occurred during previously described preparations of glycosylated Hyl derivatives, such as lactone formation, loss of side‐chain protecting groups, orthoester formation, or production of anomeric mixtures, were avoided here. Research on the enzymology of Lys hydroxylation and subsequent glycosylation, as well as the role of glycosylated Hyl in receptor recognition, will be greatly aided by the convenient and efficient synthetic methods developed here.     

Fmoe SPPS using Perloza™ beaded cellulose

Perloza? beaded cellulose was functionalised by a cyanoethylation reduction procedure to give aminopropyl Perloza. Fmoc-amino acids were anchored to aminopropyl Perloza beaded cellulose via the TFA labile 4-oxymethylphenoxyacetyl (HMPA) linker. Using Fmoc-aminoacyl-4-oxymethylphenoxyacetyl-2,4-dichloro-phenyl esters, all 20 amino acids were anchored at substitution levels ranging from 0.37 to 0.65 mmol/g. Fmoc-amino acids were also anchored using the peptide-amide linker 4-[(R,S)-1-[1-(9H-fluoren-9-yl)-methoxycarbonylamino]-(2′,4′dimethoxy-benzyl]phenoxyacetic acid. The Fmoc-aminoacyl resins were used for SPPS using Fmoc chemistry. SPPS was carried out using either an LKB Biolynx 4175 low-pressure pumped column continuous-flow peptide synthesiser or an ABI 430A automated vortexing batchwise instrument. Comparison of peptides made using each synthesiser showed little difference in quality of the crude peptides. Different Fmoc-amino acid activation methods (DIC/HOB_t/DMF, HBTU, DIC/HOB_t/DCM) were found to be equally useful with Perloza. Peptides were cleaved using TFA plus scavengers; however, the TFA-swollen resin was not readily separated from the TFA peptide solution by simple filtration. Therefore alternative cleavage workup procedures were used with Perloza. Peptides were purified by HPLC and characterised by HPLC and amino acid analysis, and in some cases by FAB-MS. Successful syntheses ranged from 5 to 34 amino acids in length. Some of the peptides were also synthesized using a polystyrene support and standardised (ABI Fastmoc?) SPPS protocols. The crude cleaved peptides from each synthesis were compared by HPLC analysis. The overall aim of our work with Perloza is synthesis of resin-bound peptide ligands for affinity chromatography and antibody generation. HPLC analysis of crude peptides showed that peptides up to 20 amino acids in length were of reasonable purity when synthesised using Perloza, thus encouraging us to continue with investigations of SPPS of resin-bound ligands.     

STABLE ISOLATED SYMMETRICAL ANHYDRIDES OF Nα-9-FLUORENYLMETHYLOXYCARBONYLAMINO ACIDS IN SOLID-PHASE PEPTIDE SYNTHESIS:

The synthesis and isolation of symmetrical anhydrides of N^α-9-fluorenylmethyloxycarbonyl (Fmoc) amino acids using water soluble carbodiimide is described. These compounds were used in a solid phase peptide synthesis of methionine-enkephalin on a p-benzyloxybenzyl ester polystyrene 1% divinylbenzene resin support. Homogeneous free pentapeptide was obtained in 42% overall yield. The Fmoc amino acid symmetrical anhydrides were stable during prolonged storage (2 years at 0°) and offer advantages over present “Fmoc solid phase” methods which use anhydrides formed in situ.     

Preparation of protected peptidyl thioester intermediates for native chemical ligation by Nα‐9‐fluorenylmethoxycarbonyl (Fmoc) chemistry: considerations of side‐chain and backbone anchoring strategies,and compatible protection for N‐terminal cysteine*,†

Abstract: Native chemical ligation has proven to be a powerful method for the synthesis of small proteins and the semisynthesis of larger ones. The essential synthetic intermediates, which are C‐terminal peptide thioesters, cannot survive the repetitive piperidine deprotection steps of N^α‐9‐fluorenylmethoxycarbonyl (Fmoc) chemistry. Therefore, peptide scientists who prefer to not use N^α‐t‐butyloxycarbonyl (Boc) chemistry need to adopt more esoteric strategies and tactics in order to integrate ligation approaches with Fmoc chemistry. In the present work, side‐chain and backbone anchoring strategies have been used to prepare the required suitably (partially) protected and/or activated peptide intermediates spanning the length of bovine pancreatic trypsin inhibitor (BPTI). Three separate strategies for managing the critical N‐terminal cysteine residue have been developed: (i) incorporation of N^α‐9‐fluorenylmethoxycarbonyl‐S‐(N‐methyl‐N‐phenylcarbamoyl)sulfenylcysteine [Fmoc‐Cys(Snm)‐OH], allowing creation of an otherwise fully protected resin‐bound intermediate with N‐terminal free Cys; (ii) incorporation of N^α‐9‐fluorenylmethoxycarbonyl‐S‐triphenylmethylcysteine [Fmoc‐Cys(Trt)‐OH], generating a stable Fmoc‐Cys(H)‐peptide upon acidolytic cleavage; and (iii) incorporation of N^α‐t‐butyloxycarbonyl‐S‐fluorenylmethylcysteine [Boc‐Cys(Fm)‐OH], generating a stable H‐Cys(Fm)‐peptide upon cleavage. In separate stages of these strategies, thioesters are established at the C‐termini by selective deprotection and coupling steps carried out while peptides remain bound to the supports. Pilot native chemical ligations were pursued directly on‐resin, as well as in solution after cleavage/purification.     

Chemical synthesis and receptor binding of catfish somatostatin: a disulfide‐bridged β‐d‐Galp‐(1→3)‐α‐d‐GalpNAc O‐glycopeptide*

Abstract: The glycopeptide hormone catfish somatostatin (somatostatin‐22) has the amino acid sequence H‐Asp‐Asn‐Thr‐Val‐Thr‐Ser‐Lys‐Pro‐Leu‐Asn‐Cys‐Met‐Asn‐Tyr‐Phe‐Trp‐Lys‐Ser‐Arg‐Thr‐Ala‐Cys‐OH; it includes a cyclic disulfide connecting the two Cys residues, and the major naturally occurring glycoform contains d ‐GalNAc and d ‐Gal O‐glycosidically linked to Thr⁵. The linear sequence was assembled smoothly starting with an Fmoc‐Cys(Trt)‐PAC‐PEG‐PS support, using stepwise Fmoc solid‐phase chemistry. In addition to the nonglycosylated peptide, two glycosylated forms of somatostatin‐22 were accessed by incorporating as building blocks, respectively, N^α‐Fmoc‐Thr(Ac₃‐α‐D‐GalNAc)‐OH and N^α‐Fmoc‐Thr(Ac₄‐β‐D‐Gal‐(1→3)‐Ac₂‐α‐D‐GalNAc)‐OH. Acidolytic deprotection/cleavage of these peptidyl‐resins with trifluoroacetic acid/scavenger cocktails gave the corresponding acetyl‐protected glycopeptides with free sulfhydryl functions. Deacetylation, by methanolysis in the presence of catalytic sodium methoxide, was followed by mild oxidation at pH 7, mediated by N^α‐dithiasuccinoyl (Dts)‐glycine, to provide the desired monomeric cyclic disulfides. The purified peptides were tested for binding affinities to a panel of cloned human somatostatin receptor subtypes; in several cases, presence of the disaccharide moiety resulted in 2‐fold tighter binding.