Peptides constrained by an aliphatic linkage between two C(alpha) sites: design, synthesis, and unexpected conformational properties of an i,(i + 4)-linked peptide.

A novel route for the synthesis of cyclic peptides constrained by an aliphatic bridge between two C(alpha)sites, using a triply orthogonal protecting group strategy, is described. The synthesis of the orthogonally protected bis-amino acid 1, via an enantioselective route utilizing the Sch?llkopf and Evans methodologies, is first described. This is then incorporated into a short, alanine-rich peptide 13, using a novel triply orthogonal protecting group strategy to couple first one, then the other, amino acid moiety in such a way that an aliphatic bridge is formed between the i and i + 4 positions. Unexpectedly, the resulting constrained peptide does not adopt a helical conformation: instead, it is shown by CD at low temperature to adopt a left-handed type II beta-turn conformation in aqueous media and a right-handed type I beta-turn conformation in TFE.     

Facile synthesis of alpha,alpha-diisobutylglycine and anchoring its derivatives onto PAL-PEG-PS resin

alpha,alpha-Diisobutylglycine has been synthesized using a Pd-mediated dialkylation of ethyl nitroacetate as a key first step. The free alphaalphaAA is N(alpha)-protected and has been applied to the assembly of conformationally constrained peptide analogues. Mixed anhydrides from BOP-Cl and Fmoc-alphaalphaAA-OH are used for anchoring alphaalphaAAs onto a trialkoxybenzyl linker on PEG-PS grafted support, upon which a beta-strand mimic with difficult sequence is assembled in a superior quality.     

An activated O --> N acyl transfer auxiliary: efficient amide-backbone substitution of hindered "difficult" peptides

Overcoming the phenomenon known as "difficult" synthetic sequences has been a major goal in solid-phase peptide synthesis for over 30 years. In this work the advantages of amide backbone-substitution in the solid-phase synthesis of "difficult" peptides are augmented by developing an activated N(alpha)()-acyl transfer auxiliary. Apart from disrupting troublesome intermolecular hydrogen-bonding networks, the primary function of the activated N(alpha)()-auxiliary was to facilitate clean and efficient acyl capture of large or beta-branched amino acids and improve acyl transfer yields to the secondary N(alpha)()-amine. We found o-hydroxyl-substituted nitrobenzyl (Hnb) groups were suitable N(alpha)()-auxiliaries for this purpose. The relative acyl transfer efficiency of the Hnb auxiliary was superior to the 2-hydroxy-4-methoxybenzyl (Hmb) auxiliary with protected amino acids of varying size. Significantly, this difference in efficiency was more pronounced between more sterically demanding amino acids. The Hnb auxiliary is readily incorporated at the N(alpha)()-amine during SPPS by reductive alkylation of its corresponding benzaldehyde derivative and conveniently removed by mild photolysis at 366 nm. The usefulness of the Hnb auxiliary for the improvement of coupling efficiencies in the chain-assembly of difficult peptides was demonstrated by the efficient Hnb-assisted Fmoc solid-phase synthesis of a known hindered difficult peptide sequence, STAT-91. This work suggests the Hnb auxiliary will significantly enhance our ability to synthesize difficult polypeptides and increases the applicability of amide-backbone substitution.     

Synthesis of novel alpha-substituted and alpha,alpha-disubstituted amino acids by rearrangement of ammonium ylides generated from metal carbenoids

[reaction: see text] A new and general four-step synthesis of protected alpha-substituted and alpha,alpha-disubstituted amino acids has been developed. The key step involves intramolecular ammonium ylide generation from a copper carbenoid with concomitant [2,3] rearrangement. The aromatic template serves as a tether, protecting group, and activating group for peptide coupling. The ylide rearrangement products can be converted into protected cyclic amino acids by ring-closing metathesis.     

Asymmetric 1,4-addition of oxazolones to nitroalkenes by bifunctional cinchona alkaloid thiourea organocatalysts: synthesis of alpha,alpha-disubstituted alpha-amino acids

An easy and simple synthetic approach to optically active alpha,alpha-quaternary alpha-amino acids using asymmetric organocatalysis is presented. The addition of oxazolones to nitroalkenes catalyzed by thiourea cinchona derivatives provides the corresponding alpha,alpha-quaternary alpha-amino acid derivatives with good yields, excellent diastereoselectivities (up to 98 % dr), and from moderate to good enantioselectivities (up to 92 % ee). The reaction can be performed on a large scale. The optically active oxazolone-nitroalkene addition products can be opened in a one-pot reaction to the corresponding ester-amide derivatives. Additional transformations are also presented, such as the synthesis of amino esters, amino acids, and transformation into 3,4-disubstituted pyrrolidin-2-ones.     

Synthesis of the core tetrasaccharide of Trypanosoma cruzi glycoinositolphospholipids: Manp(alpha1-->6)-Manp(alpha1-->4)-6-(2-aminoethylphosphonic acid)-GlcNp(alpha1-->6)-myo-Ins-1-PO4

[structure: see text] Synthesis of the core tetrasaccharide Manp(alpha1-->6)-Manp(alpha1-->4)-6-(2-aminoethylphosphonic acid)-GlcNp(alpha1-->6)-myo-Ins-1-PO4, found in glycoinositolphospholipids of Trypanosoma cruzi parasites, is described. The key building block, 6-O-(2-azido-3-O-benzyl-6-O-((2-benzyloxycarbonylaminoethyl)phosphonic acid benzyl ester)-2-deoxy-alpha-D-glucopyranosyl)-1-di-O-benzylphosphoryl-4,5-O-isopropylidene-2,3-O-(D-1,7,7-trimethyl[2,2,1]bicyclohept-6-ylidene)-D-myo-inositol, was synthesized using a partially protected glucosyl D-camphorinositolphosphate and a (2-benzyloxycarbonylaminoethyl)phosphonic acid derivative in a regioselective phosphonate esterfication. Elongation with ethyl 2-O-benzoyl-3,4,6-tri-O-benzyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzyl-1-alpha-D-thiomannopyranoside using dimethyl(methylthio)sulfonium trifluoromethanesulfonate gave a fully protected tetrasaccharide which was successfully deprotected subsequently with sodium methoxide, sodium in liquid ammonia, and aq hydrochloric acid to give title compound.     

Facile chemoselective synthesis of dehydroalanine-containing peptides

Useful methodology is described for the synthesis of dehydroalanine residues (II) within peptides. The unnatural amino acid (Se)-phenylselenocysteine (I) can be incorporated into growing peptide chains via standard peptide synthesis procedures. Subsequent oxidative elimination affords a dehydroalanine at the desired position. The oxidation conditions are mild and tolerate functionalities commonly found in peptides, including variously protected cysteine residues. To illustrate its utility, cyclic lanthionines have been synthesized by this method.     

Solid-phase synthesis of 1,5-disubstituted 2-aryliminoimidazolidines

The solid-phase synthesis of 1,5-disubstituted 2-aryliminoimidazolidines, starting from resin-bound N-acylated amino acid amides, is described. Exhaustive reduction of resin-bound acylated amino acid amides with borane-THF afforded the corresponding disecondary amines. Further reaction with arylisothiocyanates in the presence of mercuric chloride (HgCl(2)) yielded the corresponding resin-bound 1,5-disubstituted 2-aryliminoimidazolidines. Cleavage of the product from the resin using HF/anisole (95/5) for 1.5 h at 0 degrees C gave the desired products in good yield and purity. The preparation of a large combinatorial library of such compounds is also discussed.     

Ethyl 2‐benzyl‐2‐nitro‐3‐phenyl­propanoate

The title compound, C₁₈H₁₉NO₄, is the key synthetic intermediate in the preparation of α,α‐di­benzyl‐α‐amino acid (di­benzyl­glycine, Dbg), the disubstituted homologue of phenyl­alanine, following the di­alkyl­ation of ethyl nitro­acetate. The mol­ecule does not have its potential mirror symmetry in the crystal, with the two benzyl groups forming N—C—C—C torsion angles of 60.31 (13) and 79.89 (13)°.     

Stereoselective synthesis of CF(2)-substituted phosphothreonine mimetics and their incorporation into peptides using newly developed deprotection procedures

Stereoselective syntheses of all four stereoisomers of CF(2)-substituted nonhydrolyzable phosphothreonine derivatives (33, 39, and their enantiomers) and their incorporation into peptides are described herein. Key to the synthesis of these amino acids was construction of secondary phosphate-mimicking difluoromethylphosphonate units along with generation of two stereocenters. The former was achieved using a Cu(I)-mediated cross-coupling reaction of BrZnCF(2)P(O)(OEt)(2) (8) and beta-iodo-alpha,beta-unsaturated ester 12, with stereochemistry of both alpha- and beta-stereocenters being established using bornane-10,2-sultam as a chiral auxiliary. Diastereoselective hydrogenation of a chiral alpha,beta-unsaturated acylsultam (for the beta-center) (e.g., 16a) and subsequent stereoselective bromination (for the alpha-center of the threo derivative) or amination (for the alpha-center of erythro (allo) derivative) were utilized. Transesterification of the bromide to the benzyl ester followed by azide displacement of the halogen, then reduction of the resulting azide, followed by Boc-protection and finally removal of the benzyl group, afforded protected both L- and D-phosphothreonine mimetics (39 and its enantiomer). On the other hand, protected both L- and D-allo-phosphothreonine mimetics (33 and its enantiomer) were synthesized via transesterification of the above-mentioned amination product, followed by hydrogenolytic removal of the benzyl group. Key to utilization of these amino acid analogues in peptide synthesis was removal of ethyl protection from the difluoromethylphosphonate moiety. A two-step deprotection methodology, consisting of a combination of a first-step reagent [0.3 M BSTFA-TBAI in CH(2)Cl(2), BF(3).Et(2)O] followed by a second-step reagent [1 M TMSOTf-thioanisole in TFA, m-cresol, EDT] was developed for use in solid-phase protocols. A 12-residue Cdc (cell division cycle) 2-peptide 41, possessing two nonhydrolyzable phosphoamino acid mimetics (F(2)Pmab 6 and F(2)Pmp 4), was subjected to this deprotection procedure and was obtained in 25% yield based on the protected resin. The present synthetic method affords nonhydrolyzable phosphoamino acid mimetics-containing peptides in high yield without accompanying side reactions.     

Solid-phase synthesis of peptide and glycopeptide thioesters through side-chain-anchoring strategies

An efficient new strategy for the synthesis of peptide and glycopeptide thioesters is described. The method relies on the side-chain immobilization of a variety of Fmoc-amino acids, protected at their C-termini, on solid supports. Once anchored, peptides were constructed using solid-phase peptide synthesis according to the Fmoc protocol. After unmasking the C-terminal carboxylate, either thiols or amino acid thioesters were coupled to afford, after cleavage, peptide and glycopeptide thioesters in high yields. Using this method a significant proportion of the proteinogenic amino acids could be incorporated as C-terminal amino acid residues, therefore providing access to a large number of potential targets that can serve as acyl donors in subsequent ligation reactions. The utility of this methodology was exemplified in the synthesis of a 28 amino acid glycopeptide thioester, which was further elaborated to an N-terminal fragment of the glycoprotein erythropoietin (EPO) by native chemical ligation.     

Stereoselective synthesis of orthogonally protected alpha-methylnorlanthionine

[reaction: see text] As the unusual amino acid norlanthionine (nor-Lan) has previously been incorporated into cyclic peptide analogues of the ring C of lantibiotic nisin, we report here the stereoselective synthesis of the new (S,R)- and (R,R)-alpha-methylnorlanthionines (alpha-Me-nor-Lan). The orthogonally protected derivatives of these compounds have also been prepared. The key step in the synthesis of these bisamino acids was the S(N)2 opening reaction of the corresponding cyclic sulfamidates with the SH group of appropriately protected l-cysteine derivatives.     

Synthesis of nonproteinogenic amino acids to probe lantibiotic biosynthesis

The synthesis of six nonproteinogenic amino acids appropriately protected for Fmoc-based solid-phase peptide synthesis is described. These amino acids are (2S,3R)-vinylthreonine, (2S)-(E)-2-amino-5-fluoro-pent-3-enoic acid (fluoroallylglycine), (S)-beta(2)-homoserine, (S) and (R)-beta(3)-homocysteine, and (2R,3R)-methylcysteine. Once incorporated into peptides, these compounds were envisioned to serve as alternative substrates for the lantibiotic synthases that dehydrate serine and threonine residues in their peptide substrates and catalyze the subsequent intramolecular Michael-type addition of cysteines to the dehydroamino acids.     

A convenient route to N-[2-(Fmoc)aminoethyl]glycine esters and PNA oligomerization using a Bis-N-Boc nucleobase protecting group strategy

A simple and practical synthesis of the benzyl, allyl, and 4-nitrobenzyl esters of N-[2-(Fmoc)aminoethyl]glycine is described starting from the known N-(2-aminoethyl)glycine. These esters are stored as stable hydrochloride salts and were used in the synthesis of peptide nucleic acid monomers possessing bis-N-Boc-protected nucleobase moieties on the exocyclic amino groups of ethyl cytosin-1-ylacetate, ethyl adenin-9-ylacetate and ethyl (O(6)-benzylguanin-9-yl)acetate. Upon ester hydrolysis, the corresponding nucleobase acetic acids were coupled to N-[2-(Fmoc)aminoethyl]glycine benzyl ester or to N-[2-(Fmoc)aminoethyl]glycine allyl ester in order to retain the O(6) benzyl ether protecting group of guanine. The Fmoc/bis-N-Boc-protected monomers were successfully used in the Fmoc-mediated solid-phase peptide synthesis of mixed sequence 10-mer PNA oligomers and are shown to be a viable alternative to the currently most widely used Fmoc/Bhoc-protected peptide nucleic acid monomers.     

Enantioselective synthesis of an unnatural bipyridyl amino acid and its incorporation into a peptide

The synthesis of a bipyridyl amino acid, 2-amino-3-(4′-methyl-2,2′-bipyridin-4-yl) propanoic acid, is described. A short three step synthesis from commercially available 4,4′-dimethyl-2,2′-bipyridine provides the amino acid in 65% enantiomeric excess (ee). An enzyme-mediated chiral resolution increases the ee to 95% in two additional steps. The amino acid was incorporated into a 22 amino acid peptide composed predominantly of alanine. The peptide was found to be 88% α-helical in aqueous solution at 1°C by circular dichroism (CD) spectropolarimetry, indicating a high helical propensity for this amino acid. This amino acid can provide a means to incorporate a metal into structure-forming peptides.     

A strategy for the asymmetric aminohomologation of alpha, beta-dihydroxy aldehydes: application to the synthesis of the southwest tripeptide segment of echinocandin B

The synthesis of the (2S,3S,4S)-3,4-dihydroxyhomotyrosine amino acid segment, present in echinocandin B, in its activated form ready for peptide coupling is described. The key steps of the approach are the enantioselective AD reaction of 4-methoxycinnamic acid methyl ester, a completely diastereoselective [2 + 2] hydroxyketene-imine cycloaddition, and the TEMPO-assisted cycloexpansion of the resulting 3-hydroxy beta-lactam to the corresponding alpha-amino acid N-carboxy anhydride (NCA). The smooth opening of the latter upon treatment with L-Thr(OSi(t)BuPh(2))OMe and further acylation with the N-Cbz protected L-4-tert-butyldiphenylsilyloxy proline rendered the southwest portion of echinocandin B.     

Design and synthesis of alpha Gal-conjugated peptide T20 as novel antiviral agent for HIV-immunotargeting

An efficient chemo-enzymatic synthesis of alpha Gal-conjugated peptide T20 as novel HIV-immuno-targeting agent is described. The synthesis involves chemo-enzymatic preparation of maleimide-functionalized alpha Gal epitope and its chemoselective ligation with the peptide T20. The title compound contains two functional domains: the trisaccharide alpha Gal epitope that binds to human natural anti-Gal antibodies and the 36-amino acid gp41 peptide (T20) that recognizes the gp41 N-terminal ectodomain of the HIV envelope. Biological assays demonstrated that the synthetic conjugate could readily bind to natural anti-Gal antibodies (both IgG and IgM type) in normal human serum and exhibited potent anti-HIV activity even in the absence of human antibodies and complement system. The experimental data suggest that the synthetic alpha Gal-T20 might be valuable for in vivo HIV-immuno-targeting via antibody-mediated cytotoxicity and/or antibody-dependent, complement-mediated lysis of HIV particles and HIV-infected cells, thus providing an additional dimension of HIV intervention.     

Catalytic intermolecular allylic C-H alkylation

The first electrophilic Pd(II)-catalyzed allylic C H alkylation is reported, providing a novel method for formation of sp3-sp3 C C bonds directly from C H bonds. A wide range of aromatic and heteroaromatic linear (E)-alpha-nitro-arylpentenoates are obtained as single olefin isomers in excellent yields directly from terminal olefin substrates and methyl nitroacetate. The use of DMSO as a pi-acidic ligand was found to be crucial for promoting functionalization of the pi-allylPd intermediate. Products from this reaction are valuable synthetic intermediates and are readily transformed to amino esters via selective reduction and optically enriched alpha,alpha-disubstituted amino acid precursors via asymmetric conjugate addition.     

Carbohydrates as chiral templates: asymmetric ugi-synthesis of alpha-amino acids using galactosylamines as the chiral matrices

In the presence of Lewis acid catalysts O-acetyl- (1) and O-pivaloyl- (2) protected β-D-galactopyranosylamines react with aldehydes, isocyanides and carboxylic acids in Ugi-four-component-condensations to give the corresponding N-galactosyl-amino acid amide derivatives 3,5 in almost quantitative yields. Zinc chloride is the most effective Lewis acid catalyst. At 0°C or even at room temperature the (2R,β-D)-diastereomers of the amino acid derivatives 3,5 are formed with high diastereoselectivity. If the sterically more demanding O-pivaloyl galactosylamine 2 is used at -78°C to -25°C the stereoselectivity often exeeds 20:1 favouring the (2R,β-D) diastereomers 5. After one recrystallisation pure (R)-amino acid derivatives 5 are obtained in yields of 80–95%. In addition to the high yields and stereoselectivity the amino acid synthesis discribed here has the further advantage that it neither requires organometallic reagents and intermediates nor exclusion of oxygen and moisture. Two step acid hydrolysis of the N-galactosylamino acid amide derivatives 5 delivers the enantiomerically pure (R)-amino-acids 8 in high yields.     

Enzyme-labile protecting groups in peptide synthesis: development of glucose- and galactose-derived urethanes

The development of the tetra-O-acetyl-D-glucopyranosyloxycarbonyl (AGlOC) and tetra-O-acetyl-beta-D-galactopyranosyloxycarbonyl (AGalOC) protecting groups, which are fully enzyme-labile, carbohydrate-derived urethanes, is described. The protected amino acids were easily synthesized and subsequently converted into a series of model dipeptides through classical peptide couplings. Cleavage of an alpha/beta-anomeric mixture of a model AGlOC dipeptide was achieved with a "one-pot" procedure in good yield. To gain a better understanding of the enzymatic deprotection reaction, the AGalOC group was removed through a two step biotransformation (lipase catalyzed deacetylation, followed by beta-galactosidase catalyzed glycosidic bond fragmentation). Under these very mild reaction conditions (aq. buffer pH7.0, 37 degrees C), the desired N-terminal, unprotected dipeptide conjugates were obtained. The methodology was further utilized for the synthesis of an advanced tetrapeptide model system.