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.     

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.     

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.     

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.     

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.     

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.     

Immunostimulants and Toll‐like receptor ligands obtained by screening combinatorial lipopeptide collections*

Abstract: Synthetic lipopeptides carrying the head group of bacterial lipoproteins are specific ligands of Toll‐like receptors (TLR). The three fatty acids containing lipopeptides with the tripalmitoyl‐S‐glyceryl‐cysteinyl N‐terminus (Pam₃Cys) are agonists of TLR2. The structurally related lipopeptides with a head group lacking the fatty acyl residue at the amino‐terminus (Pam₂Cys) stimulate TLR2 and 6. To investigate the influence of the peptide chain of lipohexapeptides with a free N‐terminus with regard to their ability to enhance B‐cell proliferation, a randomized S‐[2,3‐bis(palmitoyloxy)‐(2RS)‐propyl]‐(R)‐cysteinyl‐pentapeptide amide collection Pam₂CysXXXXX and 5 × 19 subcollections (Pam₂CysOXXXX, Pam₂CysXOXXX, Pam₂CysXXOXX, Pam₂CysXXXOX, Pam₂CysXXXXO, O: all protein amino acids except Cys) were prepared by parallel solid‐phase synthesis. The collection represents synthetic lipopeptide analogues of the numerous bacterial lipoproteins and of mycoplasma lipoprotein. Each of the 95 subcollections is characterized by one defined and four degenerated amino acid positions thus comprising 19⁴ individual lipopeptides with free N‐terminal amino groups. High‐performance liquid chromatography electrospray mass spectrometry (HPLC–ESI‐MS) was applied for the analytical characterization of the lipohexapeptide amide subcollections and for the individual lipohexapeptide amides. The subcollections were tested for polyclonal activation of murine spleen cells, deconvolution led to highly active single S‐[2,3‐bis(palmitoyloxy)‐(2RS)‐propyl]‐(R)‐cysteinyl‐pentapeptide amides.     

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.     

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.     

Syntheses,characterization and application of cross‐linked polystyrene−ethyleneglycol acrylate resin (CLPSER) as a novel polymer support for polypeptide syntheses

Abstract: Cross‐linked polystyrene?ethyleneglycol acrylate resin (CLPSER) was developed for the solid‐phase synthesis of peptide by introducing a cross‐linker, O,O′‐bis(2‐acrylamidopropyl)polyethylene glycol₁₉₀₀ (Acr₂PEG), into polystyrene. The cross‐linker was prepared by treating acryloyl chloride with O,O′‐bis(2‐aminopropyl) polyethylene glycol₁₉₀₀[(NH₂)₂PEG] in the presence of diisopropylethylamine. The copolymer was prepared either by bulk or inverse suspension copolymerization of Acr₂PEG₁₉₀₀ and styrene using sorbitan monolaurate as the suspension stabilizer, and a mixture of ammonium peroxodisulfate and benzoyl peroxide as the radical initiators. The resin was characterized using gel‐phase ¹³C NMR, infrared (KBr) spectroscopic techniques and the morphological features of the resin were investigated using scanning electron microscopy photographs. CLPSER showed excellent swelling in a broad range of solvents and was found to be chemically inert to various reagents and solvents used in solid‐phase peptide synthesis. To demonstrate the usefulness of the new resin in polypeptide synthesis, the support was derivatized with an ‘internal reference’ amino acid (norleucine) and a handle 4‐(4‐hydroxymethyl‐3‐methoxy)butyric acid. The new resin was compared with commercial supports such as Merrifield and Sheppard resins by synthesizing an acyl carrier protein (65?74) fragment under the same experimental conditions. HPLC profiles revealed the high efficiency of the newly developed support. Resin capability in peptide synthesis was further demonstrated by the solid phase synthesis of a 25‐residue peptide from the E2/NS1 region hepatitis C viral polyprotein.     

Synthesis of carbon‐14–labelled peptides

Carbon‐14 (¹⁴C)–labelled active pharmaceutical ingredients (APIs) and investigational medicinal products (IMPs) are required for phase 0/I to phase III mass balance and micro‐dosing clinical trials. In some cases, this may involve the synthesis of ¹⁴C‐labelled peptides, and the analysis can be performed by accelerated mass spectrometry (AMS). The ¹⁴C‐peptide is typically prepared by the solid‐phase peptide synthesis (SPPS) approach using custom‐made glassware for the key coupling steps. Further modification of the purified ¹⁴C‐peptide can then be performed.     

Synthesis of 14C‐ and 14C3‐labelled Org 37462

Org 37462 (1) is the active ingredient in Orgalutran^®, an innovative product that reduces the time of treatment in in vitro fertilization from four to less than two weeks. Org 37462 is a synthetic decapeptide containing several amino acids that are unnatural in stereochemistry and/or in structure. The synthesis, starting with a ProtectingGroup‐D‐Ala‐resin, is a typical solid state synthesis. For the conduction of several metabolism studies, Org 37462 had to be labelled with carbon 14. It was decided to label D‐3‐(2‐naphthyl)alanine, the last amino acid to be coupled to the resin. We report the synthesis of [¹⁴C]‐ and [¹⁴C₃]‐Org 37462, starting from 2‐bromo‐[¹⁴C‐methyl]‐naphthalene and [¹⁴C₂]‐tert‐butyl glycinate. Copyright © 2008 John Wiley & Sons, Ltd.     

The Biological Consequences of Replacing d‐Ala in Biphalin with Amphiphilic α‐Alkylserines

Biphalin, a synthetic opioid peptide with a broad affinity for all opioid receptors (δ, μ, and κ) and high antinociceptive activity, has been under extensive study as a potential analgesic drug. This study presents the synthesis and biological properties of four new analogues of biphalin containing amphiphilic α‐alkylserines in position 2 and 2′. The incorporation of bulky α,α‐disubstituted amino acids in the peptide chain using standard peptide chemistry is often unsuccessful. We synthesized depsipeptides, and then, the desired peptides were obtained by internal O,N‐migration of the acyl residue from the hydroxyl to the amino group under mild basic conditions. The potency and selectivity of the new analogues were evaluated by a competitive receptor‐binding assay in the rat brain using [³H]DAMGO (a μ ligand) and [³H]DELT (a δ ligand). Their binding affinity is strongly dependent on the chirality of α‐alkylserine, as analogues containing (R)‐α‐alkylserines displayed higher μ receptor affinity and selectivity than those incorporating the (S)‐isomers.     

Exploring relationships between mimic configuration,peptide conformation and biological activity in indolizidin‐2‐one amino acid analogs of gramicidin S*

Abstract: Indolizidin‐2‐one amino acids (I²aas, 6S‐ and 6R‐ 1 ) possessing 6S‐ and 6R‐ring‐fusion stereochemistry were introduced into the antimicrobial peptide gramicidin S (GS) to explore the relationships between configuration, peptide conformation and biological activity. Solution‐phase and solid‐phase techniques were used to synthesize three analogs with I²aa residues in place of the d ‐Phe‐Pro residues at the turn regions of GS: [(6S)‐I²aa^{4?5,4′?5′}]GS ( 2 ), [Lys^2,2′,(6S)‐I²aa^{4?5,4′?5′}]GS ( 3 ) and [(6R)‐I²aa^{4?5,4′?5′}]GS ( 4 ). Although conformational analysis of [I²aa^{4?5,4′?5′}]GS analogs 2?4 indicated that both ring‐fusion stereoisomers of I²aa gave peptides with CD and NMR spectral data characteristic of GS, the (6S)‐I²aa analogs 2 and 3 exhibited more intense CD curve shapes, as well as greater numbers of nonsequential NOE between opposing Val and Leu residues, relative to the (6R)‐I²aa analog 4 , suggesting a greater propensity for the (6S)‐diastereomer to adopt the β‐turn/antiparallel β‐pleated sheet conformation. In measurements of antibacterial and antifungal activity, the (6S)‐I²aa analog 2 exhibited significantly better potency than the (6R)‐I²aa diastereomer 4 . Relative to GS, [(6S)‐I²aa^{4?5,4′?5′}]GS ( 2 ) exhibited usually 1/2 to 1/4 antimicrobial activity as well as 1/4 hemolytic activity. In certain cases, antimicrobial and hemolytic activities of GS were shown to be dissociated through modification at the peptide turn regions with the (6S)‐I²aa diastereomer. The synthesis and evaluation of GS analogs 2?4 has furnished new insight into the importance of ring‐fusion stereochemistry for turn mimicry by indolizidin‐2‐one amino acids as well as novel antimicrobial peptides.     

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 of enantiopure non‐natural α‐amino acids using tert‐butyl (2S)‐2‐[bis‐(tert‐butoxycarbonyl)amino]‐5‐oxopentanoate as key‐intermediate:the first synthesis of(S)‐2‐amino‐oleic acid

Abstract: A general method for the synthesis of enantiopure non‐natural α‐amino acids is described. The key intermediate tert‐butyl (2S)‐2‐[bis(tert‐butoxycarbonyl)amino]‐5‐oxopentanoate was obtained from l ‐glutamic acid after suitable protection and selective reduction of the γ‐methyl ester group by DIBALH. Wittig reaction of this chiral aldehyde with various ylides led to a variety of δ,ε‐unsaturated α‐amino acids. This methodology was applied to the synthesis of (S)‐2‐amino‐oleic acid.     

Probing opioid receptor–ligand interactions by employment of indolizidin‐9‐one amino acid as a constrained Gly2‐Gly3 surrogate in a leucine‐enkephalin mimic

Abstract: The relationship between the conformation and biological activity of Leu‐enkephalin was studied using (2S,6R,8S)‐9‐oxo‐8‐N‐(Boc)amino‐1‐azabicyclo[4.3.0]nonane‐2‐carboxylic acid [(2S,6R,8S)‐ 1 , I⁹AA] as a constrained Gly²‐Gly³ dipeptide surrogate. [I⁹AA]^2,3‐Leu‐enkephalin 12 was assembled using solid‐phase peptide synthesis on Merrifield resin with TBTU as the coupling reagent. The in vitro assays indicated that [I⁹AA]^2,3‐Leu‐enkephalin 12 exhibited affinities for the µ‐ and δ‐opioid receptors that were three orders of magnitude lower than that of Leu‐enkephalin, as well as partial agonist character for both receptors. In in vivo assays for spinal analgesia, the indolizidinone analog 12 showed significantly enhanced duration of action, indicating an increased metabolic stability. Conformational analysis was performed using NMR and CD spectroscopy. The amide temperature coefficients and ³J_NH‐CαH coupling constants for 12 could not support a hydrogen‐bonded β‐turn structure; however, its CD spectrum indicated a turn conformation. Incorporation of indolizidinone amino acid 1 into Leu‐enkephalin thus provided additional support for the importance of a turn conformation for the biological activity of the native peptide.     

Synthesis,resolution and characterization of ring substituted phenylalanines and tryptophans

Experimental protocols have been developed for the synthesis and resolution of numerous ring substituted phenylalanines and tryptophans in half mole quantities. Physical constants on these amino acids are given and their behavior on ion exchange supports (amino acid analyzer and post column ortho-phthalaldehyde derivatization) as well as that of some selected N-methylated amino acids is described. Those amino acids were then derivatized (Nα-protection with the t-butyloxycarbonyl group) for solid phase peptide synthesis.     

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.     

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.