Synthesis of cyclo-PLAI using a combination of solid- and solution-phase methods

Cyclo-PLAI was successfully synthesized using a combination of solid- and solution-phase methods. This current synthesis was found to be faster than the previously reported synthesis for the cyclic peptide. The linear precursor was synthesized on 2-chlorotrityl resin with Fmoc/t-Bu strategy. HATU/HOAt was employed as the coupling reagent in the amide bond formation on the resin. Cyclization of the linear precursor was experimented with HATU/HOAt reagents with different conditions. However, the linear precursor was best cyclized using HATU reagent in DIPEA by stirring the reaction mixture at 0?°C for 1?h and followed by stirring the reaction mixture at room temperature for 30?min, giving the cyclic product in 70% yield (calculated from the linear peptide). Both linear and cyclic products were characterized using HR-TOF-ESMS, ¹H-NMR, ¹³C-NMR, and compared with previously reported spectral data for the cyclic product.     

New heterocyclic beta-sheet ligands with peptidic recognition elements

A detailed and comprehensive overview is presented about the design, modeling, and synthesis, as well as spectroscopic characterization, of a new class of beta-sheet ligands. The characteristic feature of these compounds is a peptidic chimeric structure formed from a specific combination of aminopyrazolecarboxylic acids with naturally occurring alpha-amino acids. These hybrid peptides are designed with the aid of molecular modeling to exist mainly in an extended conformation. All their hydrogen bond donors and acceptors can be aligned at the bottom face in such a way that a perfect complementarity toward beta-sheets is obtained. Thus the aminopyrazoles impart rigidity and a highly efficient DAD sequence for the recognition of whole dipeptide fragments, whereas the natural alpha-amino acids are designed to mimick recognition sites in proteins, ultimately leading to sequence-selective protein recognition. The synthetic protocols either rely upon solution phase peptide coupling with a PMB protecting group strategy or solid-phase peptide coupling based on the Fmoc strategy, using the same protecting group. In solution, a key building block was prepared by catalytic reduction of a nitropyrazolecarboxylic acid precursor. Subsequently, it was (N-1)-protected with a PMB group, and elongated by HCTU- or T3P-assisted peptide coupling with dipeptide fragments, followed by PyClop-assisted coupling with another nitropyrazolecarboxylic acid building block. Final simultaneous deprotection of all PMB groups with hot TFA completed the high-yield protocol, which works racemization-free. After preparing a similar key building block with an Fmoc protection at N-3, we developed a strategy suitable for automated synthesis of larger hybrid ligands on a peptide synthesizer. Attachment of the first amino acid to a polystyrene resin over the Sieber amide linker is followed by an iterative sequence consisting of Fmoc deprotection with piperidine and subsequent coupling with natural alpha-amino acid via HATU/HOAt. High yields of free hybrid peptides are obtained after mild acidic cleavage from the resin, followed by deprotection of the PMB groups with hot TFA. The new aminopyrazole peptide hybrid compounds were characterized by various spectroscopic measurements including CD spectra, VT, and ROESY NMR experiments. All these accumulated data indicate the absence of any intramolecular hydrogen bonds and strongly support an extended conformation in solution, ideal for docking on to solvent-exposed beta-sheets in proteins. Initial results from aggregation tests of pathological proteins with these and related ligands look extremely promising.     

Highly efficient synthesis of sialylglycopeptides overcoming unexpected aspartimide formation during activation of Fmoc-Asn(undecadisialyloligosaccharide)-OH

Oligosaccharyl Fmoc-asparagine in which amide nitrogen of the asparagine side chain attached to the anomeric position at the reducing end, is a versatile building block and has been used for various glycopeptide synthesis using Fmoc solid-phase peptide synthesis (SPPS). We found unexpected aspartimide formation between amide nitrogen at the reducing end and α-carboxyl acid of oligosaccharyl Fmoc-asparagine during activation of α-carboxyl acid and this side reaction resulted in low coupling yields of oligosaccharyl Fmoc-asparagine with peptide-resin. This aspartimide formed efficiently using conventional coupling reagents such as PyBOP and HATU, but DEPBT afforded little of the aspartimide derivative. Activation condition using DEPBT (3.0 equiv) and DIPEA (2.0 equiv) afforded excellent yield (97%) in coupling reaction between Fmoc-Asn(CHO)-OH and peptide-resin. Based on these results, we performed a synthesis of a sialylglycopeptide, HIV-gp120 (54-63) VVLLVN(CHO)VTENF, in high yield.     

Sterically hindered C(alpha, alpha)-disubstituted alpha-amino acids: synthesis from alpha-nitroacetate and incorporation into peptides

The preparation of sterically hindered and polyfunctional C(alpha,alpha)-disubstituted alpha-amino acids (alpha alpha AAs) via alkylation of ethyl nitroacetate and transformation into derivatives ready for incorporation into peptides are described. Treatment of ethyl nitroacetate with N,N-diisopropylethylamine (DIEA) in the presence of a catalytic amount of tetraalkylammonium salt, followed by the addition of an activated alkyl halide or Michael acceptor, gives the doubly C-alkylated product in good to excellent yields. Selective nitro reduction with Zn in acetic acid or hydrogen over Raney Ni gives the corresponding amino ester that, upon saponification, can be protected with the fluorenylmethyloxycarbonyl (Fmoc) group. The first synthesis of an orthogonally protected, tetrafunctional C(alpha,alpha)-disubstituted analogue of aspartic acid, 2,2-bis(tert-butylcarboxymethyl)glycine (Bcmg), is described. Also, the sterically demanding C(alpha,alpha)-dibenzylglycine (Dbg) has been incorporated into a peptide using solid-phase synthesis. It was found that once sterically congested Dbg is at the peptide N-terminus, further chain extension becomes very difficult using uronium or phosphonium salts (PyAOP, PyAOP/HOAt, HATU). However, preformed amino acid symmetrical anhydride couples to N-terminal Dbg in almost quantitative yield in nonpolar solvent (dichloroethane-DMF, 9:1).     

末端N-Ivdde保护的氨基酸的合成

以Ivdde为保护基,对4种氨基酸(Fmoc-Dap-OH,Fmoc-Dab-OH,Fmoc-Orn-OH和Fmoc-Lys-OH)的末端氨基进行保护,分别合成了末端氨基Ivdde保护的氨基酸[Fmoc-Dap(Ivdde)-OH,Fmoc-Dab(Ivdde)-OH,Fmoc-Orn(Ivdde)-OH和Fmoc-Lys(Ivdde)-OH],其结构经1H NMR,13C NMR,IR及元素分析表征.     

Solid-Phase Total Synthesis of Bacitracin A

An efficient solid-phase method for the total synthesis of bacitracin A is reported. This work was undertaken in order to provide a general means of probing the intriguing mode of action of the bacitracins and exploring their potential for use against emerging drug-resistant pathogens. The synthetic approach to bacitracin A involves three key features: (1) linkage to the solid support through the side chain of the L-asparaginyl residue at position 12 (L-Asn(12)), (2) cyclization through amide bond formation between the alpha-carboxyl of L-Asn(12) and the side chain amino group of L-Lys(8), and (3) postcyclization addition of the N-terminal thiazoline dipeptide as a single unit. To initiate the synthesis, Fmoc L-Asp(OH)-OAllyl was attached to a PAL resin. The chain of bacitracin A was elaborated in the C-to-N direction by sequential piperidine deprotection/HBTU-mediated coupling cycles with Fmoc D-Asp(OtBu)-OH, Fmoc L-His(Trt)-OH, Fmoc D-Phe-OH, Fmoc L-Ile-OH, Fmoc D-Orn(Boc)-OH, Fmoc L-Lys(Aloc)-OH, Fmoc L-Ile-OH, Fmoc D-Glu(OtBu)-OH, and Fmoc L-Leu-OH. The allyl ester and allyl carbamate protecting groups of L-Asn(12) and L-Lys(8), respectively, were simultaneously and selectively removed by treating the peptide-resin with palladium tetrakis(triphenylphosphine), acetic acid, and triethylamine. Cyclization was effected by PyBOP/HOBT under the pseudo high-dilution conditions afforded by attachment to the solid support. After removal of the N-terminal Fmoc group, the cyclized peptide was coupled with 2-[1'(S)-(tert-butyloxycarbonylamino)-2'(R)-methylbutyl]-4(R)-carboxy-Delta(2)-thiazoline (1). The synthetic peptide was deprotected and cleaved from the solid support under acidic conditions and then purified by reverse-phase HPLC. The synthetic material exhibited an ion in the FAB-MS at m/z 1422.7, consistent with the molecular weight calculated for the parent ion of bacitracin A (MH(+) = C(73)H(84)N(10)O(23)Cl(2), 1422.7 g/mol). It was also indistinguishable from authentic bacitracin A by high-field (1)H NMR and displayed antibacterial activity equal to that of the natural product, thus confirming its identity as bacitracin A. The overall yield for the solid-phase synthesis was 24%.     

Enantioselective synthesis of stimulus-responsive amino acid via asymmetric α-amination of aldehyde

Development of a methodology to control the function of peptides and proteins is an indispensable task in the field of chemical biology and drug delivery. Recently, we reported synthesis of racemic stimulus-responsive amino acids and their application for controlling peptidyl function. In this study, we report enantioselective synthesis of a key intermediate of stimulus-responsive amino acids via asymmetric α-amination reaction of an aldehyde. The obtained chiral intermediate was converted to an Fmoc protected UV-responsive amino acid with (S)-configuration, and it was successfully incorporated into a model peptide by Fmoc solid phase peptide synthesis.     

Synthesis and structure of preorganized,C(3)-symmetric trilactam scaffolds with convergently oriented (S)-acetylthiomethyl appendages

[reaction: see text] Efficient modular synthesis of conformationally preorganized, C(3)-symmetric trilactams is reported. The allyl acetate cyclization substrate was synthesized in five steps from Garner's L-serine-derived aldehyde. After chiral ligand-mediated palladium cyclization, the resulting vinyl hydropyran was transformed into the orthogonally protected amino acids for iterative coupling. The final macrolactamization was accomplished using EDCI/HOBt or HATU/HOAt under high dilution conditions.     

A total synthesis of a highly N-methylated cyclodepsipeptide [2S,3S-Hmp]-aureobasidin L using solid-phase methods

[2S,3S-Hmp]-Aureobasidin L 2 has been successfully synthesised through a combination of solution- and solid-phase peptide synthesis. All of the Fmoc-protected residues including a depsidipeptide, Fmoc-MeVal-Hmp-OH, were prepared in solution phase. Chain elongation on chlorotrityl resin was undertaken using selected coupling reagents including HBTU/HOBt, HATU/HOAt and BTC/collidine. Cleavage of the linear depsinonapeptide was followed by cyclisation to give the desired cyclodepsipeptide.     

An artificial beta-sheet that dimerizes through parallel beta-sheet interactions

This Article introduces a simple chemical model of a beta-sheet (artificial beta-sheet) that dimerizes by parallel beta-sheet formation in chloroform solution. The artificial beta-sheet consists of two N-terminally linked peptide strands that are linked with succinic or fumaric acid and blocked along one edge with a hydrogen-bonding template composed of 5-aminoanisic acid hydrazide. The template is connected to one of the peptide strands by a turn unit composed of (S)-2-aminoadipic acid (Aaa). 1H NMR spectroscopic studies show that these artificial beta-sheets fold in CDCl3 solution to form well-defined beta-sheet structures that dimerize through parallel beta-sheet interactions. Most notably, all of these compounds show a rich network of NOEs associated with folding and dimerization. The compounds also exhibit chemical shifts and coupling constants consistent with the formation of folded dimeric beta-sheet structures. The aminoadipic acid unit shows patterns of NOEs and coupling constants consistent with a well-defined turn conformation. The present system represents a significant step toward modeling the type of parallel beta-sheet interactions that occur in protein aggregation.     

Oxyma: An Efficient Additive for Peptide Synthesis to Replace the Benzotriazole‐Based HOBt and HOAt with a Lower Risk of Explosion[1]

Oxyma [ethyl 2‐cyano‐2‐(hydroxyimino)acetate] has been tested as an additive for use in the carbodiimide approach for formation of peptide bonds. Its performance in relation to those of HOBt and HOAt, which have recently been reported to exhibit explosive properties, is reported. Oxyma displayed a remarkable capacity to inhibit racemization, together with impressive coupling efficiency in both automated and manual synthesis, superior to those of HOBt and at least comparable to those of HOAt, and surpassing the latter coupling agent in the more demanding peptide models. Stability assays showed that there was no risk of capping the resin under standard coupling conditions. Finally, calorimetry assays (DSC and ARC) showed decomposition profiles for benzotriazole‐based additives that were consistent with their reported explosivities and suggested a lower risk of explosion in the case of Oxyma.     

Macrocyclic beta-sheet peptides that mimic protein quaternary structure through intermolecular beta-sheet interactions

This paper reports the design, synthesis, and characterization of a family of cyclic peptides that mimic protein quaternary structure through beta-sheet interactions. These peptides are 54-membered-ring macrocycles comprising an extended heptapeptide beta-strand, two Hao beta-strand mimics [JACS 2000, 122, 7654] joined by one additional alpha-amino acid, and two delta-linked ornithine beta-turn mimics [JACS 2003, 125, 876]. Peptide 3a, as the representative of these cyclic peptides, contains a heptapeptide sequence (TSFTYTS) adapted from the dimerization interface of protein NuG2 [PDB ID: 1mio]. 1H NMR studies of aqueous solutions of peptide 3a show a partially folded monomer in slow exchange with a strongly folded oligomer. NOE studies clearly show that the peptide self-associates through edge-to-edge beta-sheet dimerization. Pulsed-field gradient (PFG) NMR diffusion coefficient measurements and analytical ultracentrifugation (AUC) studies establish that the oligomer is a tetramer. Collectively, these experiments suggest a model in which cyclic peptide 3a oligomerizes to form a dimer of beta-sheet dimers. In this tetrameric beta-sheet sandwich, the macrocyclic peptide 3a is folded to form a beta-sheet, the beta-sheet is dimerized through edge-to-edge interactions, and this dimer is further dimerized through hydrophobic face-to-face interactions involving the Phe and Tyr groups. Further studies of peptides 3b-3n, which are homologues of peptide 3a with 1-6 variations in the heptapeptide sequence, elucidate the importance of the heptapeptide sequence in the folding and oligomerization of this family of cyclic peptides. Studies of peptides 3b-3g show that aromatic residues across from Hao improve folding of the peptide, while studies of peptides 3h-3n indicate that hydrophobic residues at positions R3 and R5 of the heptapeptide sequence are important in oligomerization.     

An improved procedure for N- to C-directed (Inverse) solid-phase peptide synthesis

A method for solid-phase peptide synthesis in the N- to C-direction that delivers good coupling yields and a low degree of epimerization is reported. The optimized method involves the coupling, without preactivation, of the resin-bound C-terminal amino acid with excess amounts of amino acid tri-tert-butoxysilyl (Sil) esters, using HATU as coupling reagent and 2,4,6-trimethylpyridine (TMP, collidine) as a base. For the amino acids investigated, the degree of epimerization was typically 5%, except for Ser(t-Bu) which was more easily epimerized (ca. 20%). Five tripeptides (AA(1)-AA(2)-AA(3)) with different properties were used as representative model peptides in the development of the synthetic method: Asp-Leu-Glu, Leu-Ala-Phe, Glu-Asp-Val, Asp-Ser-Ile, and Asp-D-Glu-Leu. The study used different combinations of HATU and TBTU as activating agents, N, N-diisopropylethylamine (DIEA) and TMP as bases, DMF and dichloromethane as solvents, and cupric chloride as an epimerization suppressant. The epimerization of AA(2) in the coupling of AA(3) was further reduced in the presence of cupric chloride. However, the use of this reagent also resulted in a decrease in loading onto the resin and significant cleavage between AA(1) and AA(2). Experiments indicated that the observed suppressing effect of cupric chloride on epimerization in the present system merely seemed to be a result of a base-induced cleavage of the oxazolone system, the key intermediate in the epimerization process. Consequently, the cleavages were most pronounced in slow couplings. An improved synthesis of fully characterized amino acid tri-tert-butoxysilyl (Sil) ester hydrochloride building blocks is presented. The amino acid Sil esters were found to be stable as hydrochlorides but not as free bases. Although only a few peptides have been used in this study, we believe that the facile procedure devised herein should provide an attractive alternative for the solid-phase synthesis of short (six residues or less) C-terminally modified peptides, e.g., in library format.     

Strategies for the solid-phase diversification of poly-L-proline-type II peptide mimic scaffolds and peptide scaffolds through guanidinylation

A strategy for the solid-phase diversification of PPII mimic scaffolds through guanidinylation is presented. The approach involves the synthesis N-Pmc-N'-alkyl thioureas as diversification reagents. Analogues of Fmoc-Orn(Mtt)-OH can be incorporated into a growing peptide chain on Wang resin. Side chain deprotection with 1% TFA/CH2Cl2 followed by EDCI-mediated reaction of N-Pmc-N'-alkyl thioureas with the side chain amine affords arginine analogues with modified guanidine head groups. The scope, limitations, and incidental chemistry are discussed.     

Improved procedure for the synthesis of thiazolium-type peptide coupling reagents: BMTB as a new efficient reagent

An efficient scalable synthesis of 2-halothiazolium-type peptide coupling reagents has been developed. The key step is the formation of the 2-bromothiazole scaffold through cyclization of α-thiocyanato ketones with hydrogen bromide. Using this method, the new coupling reagent 2-bromo-N-methylthiazolium bromide (BMTB) was synthesized. BMTB was tested in a difficult model coupling reaction of two sterically hindered N-methylated amino acids and showed higher activity than the well-established peptide coupling reagent HATU.     

Enhanced stereoselectivity of a Cu(II) complex chiral auxiliary in the synthesis of Fmoc-L-γ-carboxyglutamic acid

L-γ-Carboxyglutamic acid (Gla) is an uncommon amino acid that binds avidly to mineral surfaces and metal ions. Herein, we report the synthesis of N-α-Fmoc-L-γ-carboxyglutamic acid γ,γ'-tert-butyl ester (Fmoc-Gla(O(t)Bu)(2)-OH), a suitably protected analogue for Fmoc-based solid-phase peptide synthesis. The residue was synthesized using a novel chiral Cu(II) complex, whose structure-based design was inspired by the blue copper protein rusticyanin. The five-coordinate complex is formed by Shiff base formation between glycine and the novel ligand (S)-2-(N-(2-methylthio)benzylprolyl)aminobenzophenone in the presence of copper. Michael addition of di-tert-butyl methylenemalonate to the α-carbon of the glycine portion of the complex occurs in a diastereoselective fashion. The resulting (S,S)-complex diastereomer can be easily purified by chromatography. Metal complex decomposition followed by Fmoc protection affords the enantiomerically pure amino acid. With the use of this novel chiral complex, the asymmetric synthesis of Fmoc-Gla(O(t)Bu)(2)-OH was completed in nine steps from thiosalicylic acid in 14.5% overall yield.     

An efficient synthetic route to glycoamino acid building blocks for glycopeptide synthesis

[reaction: see text] Chemical glycopeptide synthesis requires access to gram quantities of glycosylated amino acid building blocks. Hence, the efficiency of synthesis of such building blocks is of great importance. Here, we report a fast and highly efficient synthetic route to Fmoc-protected asparaginyl glycosides from unprotected sugars in three steps with high yields. The glycosylated amino acids were successfully incorporated into target glycopeptides 7 and 8 by standard Fmoc solid-phase peptide synthesis.     

On-resin native chemical ligation for cyclic peptide synthesis

A novel cysteine derivative, N(alpha)-trityl-S-(9H-xanthen-9-yl)-l-cysteine [Trt-Cys(Xan)-OH] has been introduced for peptide synthesis, specifically for application to a new strategy for the preparation of cyclic peptides. The following steps were carried out to synthesize the cyclic model peptide cyclo(Cys-Thr-Abu-Gly-Gly-Ala-Arg-Pro-Asp-Phe): (i). side-chain anchoring of Fmoc-Asp-OAl via its free beta-carboxyl as a p-alkoxybenzyl ester to a solid support; (ii). stepwise chain elongation of the peptide by standard Fmoc/tBu solid-phase chemistry; (iii). removal of the N-terminal Fmoc group; (iv). coupling of Trt-Cys(Xan)-OH; (v). selective Pd(0)-promoted cleavage of the C-terminal allyl ester; (vi). coupling of the C-terminal residue, i.e., H-Phe-SBzl, preactivated as a thioester; (vii). selective removal of the N(alpha)-Trt and S-Xan protecting groups under very mild acid conditions; (viii). on-resin cyclization by native chemical ligation in an aqueous milieu; and (ix). final acidolytic cleavage of the cyclic peptide from the resin. The strategy was evaluated for three supports: poly[N,N-dimethacrylamide-co-poly(ethylene glycol)] (PEGA), cross-linked ethoxylate acrylate resin (CLEAR), and poly(ethylene glycol)-polystyrene (PEG-PS) graft resin supports. For PEGA and CLEAR, the desired cyclic product was obtained in 76-86% overall yield with initial purities of approximately 70%, whereas for PEG-PS (which does not swell nearly as well in water), results were inferior. Solid-phase native chemical ligation/cyclization methodology appears to have advantages of convenience and specificity, which make it promising for further generalization.     

酸敏性PEG载体的合成及其在多肽合成中的应用

本文通过聚苯乙烯固载聚乙二醇氨基树脂与羟甲基苯氧乙酸缩合制备了具有酸敏手臂结构的对羟甲基苯氧乙酰胺ＰＥＧ树脂。在合成中改进了ＰＳ－ＰＥＧＮＨ２树脂的制备方法,提高了树脂上功能基的转化率,不必进行封闭剩余活性基的操作,就得到具有单一功能基的树脂。     

Efficient Chemical Protein Synthesis using Fmoc-Masked N-Terminal Cysteine in Peptide Thioester Segments

We report an operationally simple method to facilitate chemical protein synthesis by fully convergent and one-pot native chemical ligations utilizing the fluorenylmethyloxycarbonyl (Fmoc) moiety as an N-masking group of the N-terminal cysteine of the middle peptide thioester segment(s). The Fmoc group is stable to the harsh oxidative conditions frequently used to generate peptide thioesters from peptide hydrazide or o-aminoanilide. The ready availability of Fmoc-Cys(Trt)-OH, which is routinely used in Fmoc solid-phase peptide synthesis, where the Fmoc group is pre-installed on cysteine residue, minimizes additional steps required for the temporary protection of the N-terminal cysteinyl peptides. The Fmoc group is readily removed after ligation by short exposure (<7 min) to 20 % piperidine at pH 11 in aqueous conditions at room temperature. Subsequent native chemical ligation reactions can be performed in presence of piperidine in the same solution at pH 7.