Alpha-keto amide peptides: a synthetic strategy to resin-bound peptide isosteres for protease inhibitor screening on solid support

A synthetic strategy for the formation of resin-bound internal alpha-keto amide peptides suitable for protease inhibitor screening on solid support is presented. This general approach is based on the incorporation of alpha-keto amide building blocks during solid-phase peptide synthesis (SPPS). Such dipeptidyl building blocks were accessible using the acylcyanophosphorane methodology. The acid-labile alpha-keto carbonyl functionality was protected as a 1,3-dithiolane derivative. This protective group is fully compatible with standard SPPS reaction conditions and can be efficiently removed with N-bromosuccinimide in 10% aqueous acetone. The alpha-keto amide peptides were assembled on SPOCC-1500 resin and were characterized with high-resolution magic angle spinning (HR-MAS) NMR on bead. The methodology was evaluated and tested with a variety of building blocks containing natural and nonnatural amino acid moieties.     

Efficient solid-phase synthesis of peptide-based phosphine ligands: towards combinatorial libraries of selective transition metal catalysts

A new methodology for the solid-phase synthesis of peptide-based phosphine ligands has been developed. Solid supported peptide scaffolds possessing either primary or secondary amines were synthesised using commercially available Fmoc-protected amino acids and readily available Fmoc-protected amino aldehydes for reductive alkylation, in standard solid-phase peptide synthesis (SPPS). Phosphine moieties were introduced by phosphinomethylation of the free amines as the final solid-phase synthetic step, immediately prior to complexation with palladium(II), thus avoiding tedious protection/deprotection of the phosphine moieties during the synthesis of the ligands. The extensive use of commercial building blocks and standard SPPS makes this methodology well suited for the generation of solid-phase combinatorial libraries of novel ligands. Furthermore, it is possible to generate several different phosphine ligand libraries for every peptide scaffold library synthesised, by functionalising the scaffold libraries with different phosphine moieties. The synthesised ligands were characterised on solid support by conventional (31)P NMR spectroscopy and, cleaved from the support, as their phosphine oxides by HPLC, (1)H NMR, (31)P NMR and high resolution ESMS. Palladium(II) allyl complexes were generated from the resin bound ligands and to demonstrate their catalytic properties, palladium catalysed asymmetric allylic substitution reactions were performed. Good yields and moderate enantioselectivity was obtained for the selected combination of catalysts and substrate, but most importantly the concept of this new methodology was proven. Screening of ligand libraries should afford more selective catalysts.     

On-line synthesis of pseudopeptide library incorporating a benzodiazepinone turn mimic: biological evaluation on MC1 receptors

Alpha melanocyte stimulating hormone (alpha-MSH) is a widely distributed hormone. This tridecapeptide exhibits various biological activities mediated through different receptors. alpha-MSH binds to the melanocortin-1 receptor (MC1-R), mainly expressed in keratinocytes and melanocytes, inducing melanogenesis and anti-inflammatory processes. The central His-Phe-Arg-Trp tetrapeptide sequence of alpha-MSH is known to form a turn in the bioactive conformation. To find new potent analogs of alpha-MSH, we decided to introduce non-peptide building blocks in the alpha-MSH sequence. Molecular modeling studies showed that two amino acids of the central core sequence could be replaced by the benzodiazepinone building block without loosing the beta-turn conformation. Benzodiazepines are well-known pharmacophores exhibiting a wide scope of biological activities and are described as constrained dipeptide mimics templates. Although numerous synthetic pathways leading to benzodiazepinones have been described in literature, no methodology has 1,4-benzodiazepine-2,5-diones building blocks bearing a free carboxylic acid function and a protected amino function suitable for incorporation into peptide sequences. In this study, we report the synthesis of peptides with a benzodiazepinone moiety obtained directly during the course of solid-phase peptide synthesis (SPPS). This "on-line" strategy leads to the generation of a 54-member pseudo-peptide library of alpha-MSH analogs. After LC/MS purification, binding assays were performed on the MC1 receptor leading to the discovery of several micromolar ligands.     

Chemical synthesis of human adiponectin(19–107) bearing post-translational glycosylation

Human adiponectin(19–107), which consists of the variable region and the collagenous domain bearing post-translational modifications including glycosylation, was chemically synthesized for the first time. A glycoside of 5-hydroxylysine (Hyl) was incorporated using an α-d-glucopyranosyl-(1→2)-β-d-galactopyranosyl/Hyl-Gly building block in a benzyl-protected form by solid-phase peptide synthesis (SPPS). The molecule was assembled from four segments prepared by SPPS via native chemical ligation (NCL) and thioester methods.     

Chemoselective protection of solid-phase compatible Fmoc-phosphinic building blocks

An efficient four-step synthetic strategy able to fully discriminate hydroxyphosphinyl and carboxylic groups of Fmoc-phosphinic building blocks and related analogues has been developed. The proposed method applies selective acidic removal of the phenacyl (Pac) group from the hydroxyphosphinyl functionality and protection by the 1-adamantyl (Ad) group. Reductive removal of the Pac group from the carboxylic functionality leads to Fmoc-protected phosphinic pseudodipeptidic units suitable for phosphinic peptide and library development using solid-phase peptide synthesis (SPPS).     

1-Ethoxyethylidene, a new group for the stepwise SPPS of aminooxyacetic acid containing peptides

For more than a decade, the oxime ether ligation has proven to be one of the most efficient technique for the preparation of various peptide conjugates. However, despite numerous reports, the preparation of aminooxy-containing peptides is still hampered by N-overacylation of the NH-O function either during its incorporation or through the peptide-chain elongation. This restricts the introduction of protected-NH-O function at the last acylation step and prevents the use of standard solid-phase peptide synthesis (SPPS) procedures for the preparation of more complex aminooxy-peptides. We have studied the coupling of modified Fmoc-lysine containing either N-Boc- or N,N'-bis-Boc-protected aminooxyacetic acids (Aoa) during the elongation of the peptide chain and found that none of them is adequate. To circumvent this limitation, we propose to protect the Aoa moiety with a 1-ethoxyethylidene group (Eei) to provide 2-(1-ethoxyethylideneaminooxy)acetic acid building block. We showed that the Eei group is fully compatible with standard SPPS conditions and safely allows the multiple incorporation of the aminooxy functionality into the growing peptide. Since Eei-protected Aoa remains as flexible as normal amino acids in peptide synthesis, it may become the rule for the straightforward preparation of aminooxy peptides.     

Caged O-phosphorothioyl amino acids as building blocks for Fmoc-based solid phase peptide synthesis

The synthesis of 1-(2-nitrophenylethyl) caged O-phosphorothioylserine, -threonine, and -tyrosine derivatives is reported. These amino acid building blocks can be directly incorporated into peptides by Fmoc-based solid phase synthesis as their pentafluorophenyl esters or as symmetric anhydrides. Upon irradiation with UV light, the thiophosphate group, representing a hydrolysis resistant phosphate analog, is revealed.     

Solid-supported synthesis of cryptand-like macrobicyclic peptides

A straightforward method for the solid-supported synthesis of cryptand-like bicyclic peptides (1-5) on a backbone amide linker has been described. For the branching, two novel easily available building blocks, viz. N-(4-methoxytrityl)-N-(2-nitrobenzenesulfonyl)-protected N,N-bis(2-aminoethyl)-beta-alanine (6) and N-(9-fluorenylmethoxycarbonyl) protected iminodiacetic acid monoallyl ester (7), have been employed. The key steps of the synthesis are as follows: (i) stepwise coupling of one amino acid and 6 to the secondary amino group of the linker; (ii) removal of the 2-nitrobenzenesulfonyl group and SPPS by the Fmoc chemistry, using 7 as the penultimate and tert-butoxycarbonyl (Boc) protected glycine as the last amino acid; (iii) removal of the 4-methoxytrityl protection and subsequent SPPS by the Fmoc chemistry; (iv) removal of the allyl and Fmoc groups, followed by cyclization; and (v) removal of the Boc and tert-butyl groups, followed by cyclization. Final cleavage from the support and removal of benzyl-derived protecting groups gives the desired bicyclic products.     

An amine-derivatized, DOTA-loaded polymeric support for Fmoc solid phase peptide synthesis

An amine-derivatized DOTA has been used to modify the surface of a polymeric support for conventional solid phase peptide synthesis (SPPS) following standard Fmoc chemistry methods. This methodology was used to synthesize a peptide-DOTA conjugate that was demonstrated to be a PARACEST MRI contrast agent. Therefore, this synthesis methodology can facilitate Fmoc SPPS of molecular imaging contrast agents.     

Synthesis and properties of azoles and their derivatives

A method is proposed for the synthesis of 1-(2-cyanoethyl)-2-substituted 2-imidazolines; the method is based on reaction of N-(2-cyanoethyl)ethylenediamine with the hydrochlorides of methyl imino esters of carboxylic acids.See [1] for communication XVI.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 12, pp. 1674–1675, December, 1973.     

Preparation of protected syn-alpha,beta-dialkyl beta-amino acids that contain polar side chain functionality

We report the synthesis of syn-alpha,beta-dialkyl beta-amino acid derivatives suitably protected for solid-phase synthesis that give rise to residues containing positively charged lysine-like side chains. These amino acids, as well as syn-alpha,beta-dialkyl beta-amino acids that contain diverse hydrophobic side chains, are prepared in good de and ee. The key step in this route involves Davies's protocol for the conjugate addition of a chiral lithium amide to alpha,beta-unsaturated tert-butyl esters (Davies, S. G.; Ichihara, O.; Walters, I. A. S. J. Chem. Soc., Perkin Trans. 1 1994, 9, 1141). syn-alpha,beta-Dialkyl beta-amino acids are interesting building blocks because of their sheet-forming propensity and because of their presence in bioactive compounds.     

Amino acid building blocks for efficient Fmoc solid-phase synthesis of peptides adenylylated at serine or threonine

The first straightforward building block based (non-interassembly) synthesis of peptides containing adenylylated serine and threonine residues is described. Key features include final global acidolytic protective group removal as well as full compatibility with standard Fmoc solid-phase peptide synthesis (SPPS). The described Thr-AMP SPPS-building block has been employed in the synthesis of the Thr-adenylylated sequence of human GTPase CDC42 (Ac-SEYVP-T(AMP)-VFDNYGC-NH(2)). Further, we demonstrate proof-of-concept for the synthesis of an Ser-adenylylated peptide (Ac-GSGA-S(AMP)-AGSGC-NH(2)) from the corresponding adenylylated serine building block.     

Solution-phase synthesis of a combinatorial library of 3-[4-(coumarin-3-yl)-1,3-thiazol-2-ylcarbamoyl]propanoic acid amides

The parallel solution-phase synthesis of a new combinatorial library of 3-[4-(R1-coumarin-3-yl)-1,3-thiazol-2-ylcarbamoyl]propanoic acid amides 9 has been developed. The synthesis involves two steps: 1) the synthesis of core building blocks - 3- [4-(coumarin-3-yl)-1,3-thiazol-2-ylcarbamoyl]propanoic acids, 6 - by the reaction of 3-(omega-bromacetyl)coumarins 1 with 3-amino(thioxo)methylcarbamoylpropanoic acid (5); 2) the synthesis of the corresponding 3-[4-(coumarin-3-yl)-1,3-thiazol-2-yl- carbamoyl]propanoic acids amides 9 using 1,1'-carbonyldimidazole as a coupling reagent. The advantages of the method compared to existing ones are discussed.     

Rational synthesis of beta-substituted chlorin building blocks

Chlorins bearing synthetic handles at specific sites about the perimeter of the macrocycle constitute valuable building blocks. We previously developed methodology for preparing meso-substituted chlorin building blocks and now present methodology for preparing several complementary beta-substituted chlorin building blocks. The chlorins bear one or two beta substituents, one meso substituent, a geminal dimethyl group to lock in the chlorin hydrogenation level, and no flanking meso and beta substituents. The synthesis involves convergent joining of an Eastern half and a Western half. New routes have been developed to two beta-substituted bromo-dipyrromethane monocarbinols (Eastern halves). A new beta-substituted Western half was prepared following the method for preparing an unsubstituted Western half (3,3-dimethyl-2,3-dihydrodipyrrin). Chlorin formation is achieved by a two-flask process of acid-catalyzed condensation followed by metal-mediated oxidative cyclization. beta-Substituted chlorins have been prepared in 18-24% yield bearing a 4-iodophenyl group at the 8-position, a 4-iodophenyl group or a 4-[2-(trimethylsilyl)ethynyl]phenyl group at the 12-position, and a 4-iodophenyl group and a 4-[2-(trimethylsilyl)ethynyl]phenyl group at diametrically opposed beta-positions (2, 12). The latter building block makes possible the stepwise construction of linear multi-chlorin architectures. The chlorins exhibit typical absorption and fluorescence spectra. A systematic shift in the absorption maximum (637-655 nm for the free base chlorins, 606-628 nm for the zinc chlorins) and intensity of the chlorin Q(y)() band (epsilon up to 79 000 M(-)(1) cm(-)(1)) is observed depending on the location of the substituents. The characteristic spectral features and location of substituents in defined positions make these chlorins well suited for a variety of applications in biomimetic and materials chemistry.     

Synthetic model of the phosphate binding protein: solid-state structure and solution-phase anion binding properties of a large oligopyrrolic macrocycle

The macrocyclic receptors 4-6 were synthesized via the anion-templated condensation of appropriately chosen dialdehyde and diamine building blocks. Whereas all three products could be obtained directly via the appropriate choice of reaction conditions, the larger [3+3] product, 6, which incorporates three of each precursor subunit, could also be obtained conveniently via an indirect procedure involving ring expansion of the smaller [2+2] macrocycle 4. As detailed earlier (Sessler, J. L.; Katayev, E. A.; Pantos, G. D.; Reshetova, M. D.; Khrustalev, V. N.; Lynch, V. M.; Ustynyuk, Y. A. Angew. Chem. 2005, 117, 7552-7556; Angew. Chem., Int. Ed. 2005, 44, 7386-7390), this ring expansion occurs under thermodynamic control in the presence of HSO4- and H2PO4- anions in acetonitrile solution and serves to effect the conversion of 4 to 6. An analysis of the X-ray crystal structure of complex 6H22+.HPO42- revealed a strong resemblance to the active site of the phosphate binding protein (PBP) with similar structural analogies being drawn between the active site of the sulfate binding protein (SBP) and the corresponding hydrogensulfate anion complex. In both cases, the anions are bound in a 1:1 fashion in the solid state through a complementary hydrogen bond network involving both the receptor 6 and the anions. UV-vis spectroscopic titrations provide support for the conclusion that macrocycle 6 binds the hydrogensulfate and dihydrogenphosphate anion (studied as the corresponding tetrabutylammonium salts) with high selectivity and affinity in acetonitrile (log Ka for the first binding interaction approaching 7), albeit with different receptor-to-anion binding stoichiometries (1:1 vs 1:3 for HSO4- and H2PO4-, respectively).     

Probing slow dynamics in high molecular weight proteins by methyl-TROSY NMR spectroscopy: application to a 723-residue enzyme

A new CPMG-based multiple quantum relaxation dispersion experiment is presented for measuring millisecond dynamic processes at side-chain methyl positions in high molecular weight proteins. The experiment benefits from a methyl-TROSY effect in which cancellation of intramethyl dipole fields occurs, leading to methyl (13)C-(1)H correlation spectra of high sensitivity and resolution (Tugarinov, V.; Hwang, P. M.; Ollerenshaw, J. E.; Kay, L. E. J. Am. Chem. Soc. 2003, 125, 10420-10428). The utility of the methodology is illustrated with an application to a highly deuterated, methyl-protonated sample of malate synthase G, an 82 kDa enzyme consisting of a single polypeptide chain. A comparison of the sensitivity obtained using the present approach relative to existing HSQC-type (13)C single quantum dispersion experiments shows a gain of a factor of 5.4 on average, significantly increasing the range of applications for this methodology.     

Design and synthesis of a new class of arginine analogues with an improved anion binding site in the side chain

Replacing the guanidinium group in arginine (1) by a guanidiniocarbonyl pyrrole moiety provides a new class of artificial amino acids (2), that can be used as building blocks in standard solid phase peptide synthesis.     

N-triazinylammonium tetrafluoroborates. A new generation of efficient coupling reagents useful for peptide synthesis

A new generation of triazine-based coupling reagents (TBCRs), designed according to the concept of "superactive esters", was obtained by treatment of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium (DMTMM) chloride with lithium or silver tetrafluoroborate. The structure of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate was confirmed by X-ray diffraction. Activation of carboxylic acids by using this reagent proceeds via triazine "superactive ester". The coupling reagent was successfully used for the synthesis of Z-, Boc-, and Fmoc-protected dipeptides derived from natural and unnatural sterically hindered amino acids and for fragment condensation, in 80-100% yield and with high enantiomeric purity. The manual SPPS of the ACP(65-74) peptide fragment (H-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-OH) proceeded significantly faster than with TBTU or HATU, as well as the automated SPPS of the same fragment gave a purer product than by using TBTU or PyBOP. The reagent was also demonstrated to be efficient in on-resin head-to-tail cyclization of constrained cyclopeptides, in SPPS synthesis of Aib peptides, and in the synthesis of esters from appropriate acids, alcohols, and phenols. The high efficiency and versatility of this new generation of TBCRs confirm, for the first time, the usefulness of the concept of "superactive esters" in rational design of the structure of coupling reagents.     

Rapid Assembly of Oligosaccharides: Total Synthesis of a Glycosylphosphatidylinositol Anchor of Trypanosoma brucei

Six building blocks, six reaction steps : The recently developed innovative methodology facilitated the convergent synthesis of the complex oligosaccharide core 1 (shown here with protecting groups) for the total synthesis of a glycosylphosphatidylinositol (GPI) anchor. The key factors are the tuning of the reactivity of the building blocks by using 1,2-diacetal protecting groups and the desymmetrization of glycerol and myo-inositol with a chiral bis(dihydropyran).     

Convenient Syntheses of Some C4 Chiral Building Blocks Starting From (S)-Malic Acid

In the field of chiral technology, the synthesis of chiral intermediates and chiral building blocks occupies an important position. Chiral building blocks bearing double and / or multiple functionalities is particularly useful for the synthesis of chiral pharmaceuticals and chiral agrochemicals. In the recent years, we have been engaged in the development of synthetic methodology based on (S)-malic acid1-s. In these studies, malimide 2, easily accessible from (S)-malic acid, was shown to be a useful multifunctional building block in the asymmetric synthesis of natural products and chiral drugs (Scheme 1).