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 of different types of dipeptide building units containing N‐ or C‐terminal arginine for the assembly of backbone cyclic peptides*

Abstract: Different types of dipeptide building units containing N‐ or C‐terminal arginine were prepared for synthesis of the backbone cyclic analogues of the peptide hormone bradykinin (BK: Arg‐Pro‐Pro‐Gly‐Phe‐Ser‐Pro‐Phe‐Arg). For cyclization in the N‐terminal sequence N‐carboxyalkyl and N‐aminoalkyl functionalized dipeptide building units were synthesized. In order to avoid lactam formation during the condensation of the N‐terminal arginine to the N‐alkylated amino acids at position 2, the guanidino function has to be deprotected. The best results were obtained by coupling Z‐Arg(Z)₂‐OH with TFFH/collidine in DCM. Another dipeptide building unit with an acylated reduced peptide bond containing C‐terminal arginine was prepared to synthesize BK‐analogues with backbone cyclization in theC‐terminus. To achieve complete condensation to the resin and to avoid side reactions during activation of the arginine residue, this dipeptide unit was formed on a hydroxycrotonic acid linker. HYCRAM^? technology was applied using the Boc‐Arg(Alloc)₂‐OH derivative and the Fmoc group to protect the aminoalkyl function. The reduced peptide bond was prepared by reductive alkylation of the arginine derivative with the Boc‐protected amino aldehyde, derived from Boc‐Phe‐OH. The best results for condensation of the branching chain to the reduced peptide bond were obtained using mixed anhydrides. Both types of dipeptide building units can be used in solid‐phase synthesis in the same manner as amino acid derivatives.     

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.     

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.     

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.     

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.     

Acid-labile anchoring linkages for solid phase synthesis of C-terminal asparagine peptides using the Fmoc strategy

Two acid-labile substituted benzylamine type anchoring linkages, 4-benzoxy-2,6-dimethoxybenzylamine and 2-benzoxy-4,6-dimethoxybenzylamine, for solid phase synthesis of peptide amides were prepared. The N^a-9-fluorenylmethyloxycarbonyl (Fmoc) amino acids could be easily attached to the resins with DCC/HOBt (loading 0.5–0.6 mmol/g resin). After final removal of the N^a-protecting groups, treatment with TFA (50–95%) yielded amino acid and peptide amides in high purity. As we could show for the synthesis of thymulin (FTS, pGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn), these two resins with anchoring linkages are well suited for the synthesis of C-terminal Asn peptides using protected aspartic acid derivative as starting material.     

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.     

SOLID-PHASE PEPTIDE SYNTHESIS USING MILD BASE CLEAVAGE OF NαFLUORENYLMETHYLOXYCARBONYLAMINO ACIDS,EXEMPLIFIED BY A SYNTHESIS OF DIHYDROSOMATOSTATIN

N ^α-9-Fluorenylmethyloxycarbonyl (Fmoc) amino acids will be of advantage in solid phase peptide synthesis. The Fmoc-group is quantitatively cleaved by mild base (piperidine). This permits the use of tert-butyl-type side chain blocking and of peptide-to-resin linkage cleavable by mild acidolysis. Side reactions arising from repetitive acid deprotection and final HF cleavage in contemporary solid phase synthesis are avoided. Fully bioactive and homogeneous dihydrosomatostatin was obtained in 53% overall yield.     

Synthesis and biological activities of new side chain and backbone cyclic bradykinin analogues

Abstract: A series of conformationally constrained cyclic analogues of the peptide hormone bradykinin (BK, Arg‐Pro‐Pro‐Gly‐Phe‐Ser‐Pro‐Phe‐Arg) was synthesized to check different turned structures proposed for the bioactive conformation of BK agonists and antagonists. Cycles differing in the size and direction of the lactam bridge were performed at the C‐ and N‐terminal sequences of the molecule. Glutamic acid and lysine were introduced into the native BK sequence at different positions for cyclization through their side chains. Backbone cyclic analogues were synthesized by incorporation of N‐carboxy alkylated and N‐amino alkylated amino acids into the peptide chain. Although the coupling of Fmoc‐glycine to the N‐alkylated phenylalanine derivatives was effected with DIC/HOAt in SPPS, the dipeptide building units with more bulky amino acids were pre‐built in solution. For backbone cyclization at the C‐terminus an alternative building unit with an acylated reduced peptide bond was preformed in solution. Both types of building units were handled in the SPPS in the same manner as amino acids. The agonistic and antagonistic activities of the cyclic BK analogues were determined in rat uterus (RUT) and guinea‐pig ileum (GPI) assays. Additionally, the potentiation of the BK‐induced effects was examined. Among the series of cyclic BK agonists only compound 3 with backbone cyclization between positions 2 and 5 shows a significant agonistic activity on RUT. To study the influence of intramolecular ring closure we used an antagonistic analogue with weak activity, [d ‐Phe⁷]‐BK. Side chain as well as backbone cyclization in the N‐terminus of [d ‐Phe⁷]‐BK resulted in analogues with moderate antagonistic activity on RUT. Also, compound 18 in which a lactam bridge between positions 6 and 9 was achieved via an acylated reduced peptide bond has moderate antagonistic activity on RUT. These results support the hypothesis of turn structures in both parts of the molecule as a requirement for BK antagonism. Certain active and inactive agonists and antagonists are able to potentiate the bradykinin‐induced contraction of guinea‐pig ileum.     

Use of the excluded protecting group (EPG) method for peptide synthesis

Abstract: The excluded protecting group (EPG) method has been used for the solution synthesis of several peptides including Merrifield's Model Tetrapeptide, linear antamanide and an analogue of magainin‐1, [Ala¹⁹, Asn²²]magainin‐1. In the approach reported, the C‐terminal amino acid is esterified to the 2‐position of cholestane as the [2s,3s]iodohydrin ester and the penultimate amino acid added to the aminoacyl‐steroid as the Fmoc‐pentafluorophenyl‐ester. The Fmoc group is removed with Et₂NH/DMF (～15% v/v) and, after evaporation to ～10 mL, the solution chromatographed on Sephadex LH‐20 in DMF. The dipeptidyl‐steroid elutes as the free amine well separated from other reaction mixture components. Fractions containing the dipeptide, as determined by counting and TLC, are pooled and reacted with the next Fmoc‐amino acid‐pentafluorophenyl ester in the sequence. Repetition of the deprotection/purification/reaction cycle yields the fully protected peptide.On completion of the synthesis, the cholestane iodohydrin ester is selectively removed by treatment with Zn°/AcOH to yield the peptide with intact α‐amino and side chain protecting groups. Global deprotection is achieved with HF. All intermediates from the syntheses reported were characterized. The magainin analogue was shown to have full biologic activity. The Fmoc iodohydrin esters of 16 of the 20 proteogenic amino acids have been prepared and characterized for use as the C‐terminal amino acids in other EPG syntheses.     

A concise methodology for the stereoselective synthesis of O-glycosylated amino acid building blocks: complete 1HNMR assignments and their application in solid-phase glycopeptide synthesis

A facile strategy for the stereoselective synthesis of suitably protected O-glycosylated amino acid building blocks, namely, N^α-Fmoc-Ser-[Ac₄-β-d -Gal-(1-3)-Ac₂α or β-d -GalN₃]-OPfp and N^α-Fmoc-Thr-[Ac₄-β-d -Gal-(1-3)-Ac₂-α or β-d -GalN₃]-OPfp is described. What is new and novel in this report is that Koenigs-Knorr type glycosylation of an aglycon serine/threonine derivative (i.e. N^α-Fmoc-Ser-OPfp or N^α-Fmoc-Thr-OPfp) with protected β-d -Gal(1-3)-d -GalN₃ synthon mediated by silver salts resulted in only α-and/or β-isomers in excellent yields under two different reaction conditions. The subtle differences in stereoselectivity were demonstrated clearly when glycosylation was carried out using only AgClO₄ at -40°C which afforded α-isomer in a quantitative yield (α:β= 5:1). On the other hand, the β-isomer was formed exclusively when the reaction was performed in the presence of Ag₂CO₃AgClO₄ at room temperature. A complete assignment of ¹H resonances to individual sugar ring protons and the characteristic anomeric α-1H and β-1H in Ac₄Galβ(1-3)Ac₂GalN₃α and/or β linked to Ser/Thr building blocks was accomplished unequivocally by two-dimensional double-quantum filtered correlated spectroscopy and nuclear Overhauser enhancement and exchange spectroscopy NMR experiments. An unambiguous structural characterization and documentation of chemical shifts, including the coupling constants for all the protons of the aforementioned a- and p-isomers of the O-glycosylated amino acid building blocks carrying protected β-d -Gal(1-3)-d -GalN₃, could serve as a template in elucidating the three-dimensional structure of glycoproteins. The synthetic utility of the building blocks and versatility of the strategy was exemplified in the construction of human salivary mucin (MUC7)-derived, O-linked glycopeptides with varied degrees of glycosylation by solid-phase Fmoc chemistry. Fmoc/tert-butyl-based protecting groups were used for the peptidic     

Protease‐catalyzed fragment condensation via substrate mimetic strategy: a useful combination of solid‐phase peptide synthesis with enzymatic methods

Abstract: The concept of substrate mimetic strategy represents a new powerful method in the field of enzymatic peptide synthesis. This strategy takes advantage of the shift in thesite‐specific amino acid moiety from the acyl residue to the ester‐leaving group of the carboxyl component enabling acylation of the enzyme by nonspecific acyl residues. As a result, peptide bond formation occurs independently of the primary specificity of proteases. Moreover, because of the coupling of nonspecific acyl residues, the newly formed peptide bond is not subject to secondary hydrolysis achieving irreversible peptide synthesis. Here, we report the combination of solid‐phase peptide synthesis with substrate mimetic‐mediated enzymatic peptide fragment condensations. First, the utility of the oxime resin strategy for the synthesis of peptide fragments in the form of substrate mimetics esterified as 4‐guanidinophenyl‐, phenyl‐ and mercaptopropionic acid esters was investigated. The study was completed by using the resulting N^α‐protected peptide esters as acyl donors in trypsin‐, α‐chymotrypsin‐ and V8 protease‐catalyzed fragment condensations.     

Amino acid–azetidine chimeras: synthesis of enantiopure 3‐substituted azetidine‐2‐carboxylic acids

Abstract: Azetidine‐2‐carboxylic acid (Aze) analogs possessing various heteroatomic side chains at the 3‐position have been synthesized by modification of 1‐9‐(9‐phenylfluorenyl) (PhF)‐3‐allyl‐Aze tert‐butyl ester (2S,3S)‐ 1 . 3‐Allyl‐Aze 1 was synthesized by regioselective allylation of α‐tert‐butyl β‐methyl N‐(PhF)aspartate 13 , followed by selective ω‐carboxylate reduction, tosylation, and intramolecular N‐alkylation. Removal of the PhF group and olefin reduction by hydrogenation followed by Fmoc protection produced nor‐leucine–Aze chimera 2 . Regioselective olefin hydroboration of (2S,3S)‐ 1 produced primary alcohol 23 , which was protected as a silyl ether, hydrogenated and N‐protected to give 1‐Fmoc‐3‐hydroxypropyl‐Aze 26 . Enantiopure (2S,3S)‐3‐(3‐azidopropyl)‐1‐Fmoc‐azetidine‐2‐carboxylic acid tert‐butyl ester 3 was prepared as a Lys–Aze chimera by activation of 3‐hydroxypropyl–Aze 26 as a methanesulfonate and displacement with sodium azide. Moreover, orthogonally protected azetidine dicarboxylic acid 4 was synthesized as an α‐aminoadipate–Aze chimera by oxidation of alcohol 26 . These orthogonally protected amino acid–Aze chimeras are designed to serve as tools for studying the influence of conformation on peptide activity.     

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.     

Zinc‐promoted simple synthesis of oligomer‐freeNα‐Fmoc‐amino acids using Fmoc‐Cl as an acylating agent under neutral conditions

Abstract: A range of N^α‐Fmoc‐protected amino acids, including those that contain t‐butyl moiety, have been synthesized by employing Fmoc‐Cl utilizing the activated, commercial zinc dust‐promoted synthesis of carbamates under neutral conditions.A general procedure is described that circumvents the oligomerization side reaction normally noticed in Schotten–Baumann conditions. It is a simple, convenient and clean method. Thus, Fmoc‐amino acids are obtained in high yield (85–92%) and purity as checked by thin‐layer chromatography, high‐performance liquid chromatography and other physical methods.     

Insect neuropeptide antagonist. Part II. Synthesis and biological activity of backbone cyclic and precyclic PBAN antagonists

Abstract: A new approach for the design and synthesis of pheromone biosynthesis activating neuropeptide (PBAN) agonists and antagonists using the backbone cyclization and cycloscan concepts is described. Two backbone cyclic (BBC) libraries were synthesized: library I (Ser library) was based on the active C‐terminal hexapeptide sequence Tyr‐Phe‐Ser‐Pro‐Arg‐Leu‐NH₂ of PBAN1‐33NH₂; whereas library II (d ‐Phe library) was based on the sequence of the PBAN lead linear antagonist Arg‐Tyr‐Phe‐d ‐Phe‐Pro‐Arg‐Leu‐NH₂. In both libraries the Pro residue was replaced by the BBC building unit N^α‐(ω‐aminoalkyl) Gly having various lengths of alkyl chain. The peptides of the two libraries were tested for agonistic and antagonistic activity. Four precyclic peptides based on two of the BBC antagonists were also synthesized; their activity revealed that a negative charge at the N‐terminus of the peptide abolished antagonistic activity. We also describe the use of the reagent SiCl₃I for selective deprotection of the Boc group from the building unit prior to on‐resin amino‐end to backbone‐nitrogen (AE‐BN) cyclization, during solid‐phase synthesis with Fmoc chemistry.     

Facile synthesis of Nα‐protected‐l‐α,γ‐diaminobutyric acids mediated by polymer‐supported hypervalent iodine reagent in water

Abstract: Hofmann rearrangement of N^α‐Boc‐l ‐Gln‐OH mediated by a polymer‐supported hypervalent iodine reagent poly[(4‐diacetoxyiodo)styrene] (PSDIB) in water afforded N^α‐Boc‐l ‐α,γ‐diaminobutyric acid (Boc‐Dab‐OH, 1 ) in 87% yield. N^α‐Z‐derivative (Z‐Dab‐OH, 2 ) was prepared with PSDIB in 83% yield. Since the reaction of N^α‐Fmoc‐Gln‐OH by this procedure did not proceed because of the insolubility of Fmoc‐Gln‐OH in aqueous media, we synthesized Fmoc‐Dab(Boc)‐OH ( 5 ) from 2 in 54% yield. Polymyxin B heptapeptide (PMBH) which contains four Dab residues was successfully synthesized in a solution‐phase synthesis.     

A convenient incorporation of conformationally constrained 5,5‐dimethylproline into the ribonuclease A 89–124 sequence by condensation of synthetic peptide fragments

Abstract: The presence of l ‐5,5‐dimethylproline (dmP) within an amino acid sequence results in the formation of an X‐dmP peptide bond predominantly locked in a cis conformation. However, the common use of this unnatural amino acid has been hampered by the difficulty of the economical incorporation of the dmP residue into longer peptide segments due to the steric hindrance imposed by the dimethyl moieties. Here, we describe synthesis of the C‐terminal 36‐residue peptide, corresponding to the 89–124 sequence of bovine pancreatic ribonuclease A (RNase A), in which dmP is incorporated as a substitute for Pro⁹³. The peptide was assembled by condensation of protected 5‐ and 31‐residue peptide fragments, which were synthesized by solid‐phase peptide methodology using fluorenylmethyloxycarbonyl chemistry. We focused on optimizing the synthesis of the Fmoc‐Ser(^tBu)‐Ser(^tBu)‐Lys(Boc)‐Tyr(^tBu)‐dmP‐OH pentapeptide (residues 89–93) with efficient acylation of the sterically hindered dmP residue. In a comparative study, the reagent O‐(7‐azabenzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate was found to be superior to bromo‐tris‐pyrrolidino‐phosphonium hexafluorophosphate and tetramethylfluoroformamidinium hexafluorophosphate for the synthesis of the ‐Tyr(^tBu)‐dmP‐ peptide bond in solution as well as on a resin.