No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Synthesis of D-lysine8-cyclosporine A. Further characterization of BOP-Cl in the 2-7 hexapeptide fragment synthesis
Abstract
The coupling reagent N,N-bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl) has been used to synthesize the 2-7 hexapeptide fragment of cyclosporine A. The reagent proved useful in four of the five coupling steps with yields ranging from 78 to 92%. Overall, this BOP-Cl mediated synthesis proved to be more difficult than the previously described 8-11 tetrapeptide fragment synthesis (J. Org. Chem. 1986, 51, 3350). Prudent choices of both protecting groups and reaction conditions were required. The best approach, which employed Pmz-N-protection of the 5-7 tripeptide and Fmoc-N-protection of the 4-7 tetrapeptide gave the fully protected hexapeptide in 51% overall yield for the coupling steps. The 2-7 fragment, thus obtained, was used to prepare D-lysine8-cyclosporine A. This analogue, Containing D-lysine at the 8-position of cyclosporine A, is useful for isolating and characterizing putative receptors of cyclosporine A.
... Our synthesis is shown in Scheme 1, and is adapted from Rich's total synthesis of [D-Lys] 8 CsA, 14 combined with regioselective ring-opening methodology first reported by Seebach 15 CsA (1) was first treated with Ac 2 O to protect the hydroxyl group of residue 1. 17 The resulting acetate (5) was then treated with 1.0 equivalent of Lawesson's reagent, which regioselectively reacted with the sterically accessible amide carbonyl groups of residues 4 and 7 to give a statistical mixture of 7-thioamide 6, 4-thioamide 7, 4,7bisthioamide 8, and unreacted 5, all of which were partially separated by chromatography. 15,16 A mixture of 6 and 8 was then treated with benzyl bromide and DBU to give the corresponding benzyl thioamidates; 16 the 4,7-bisbenzyl thioamidate (10) was considerably less polar than the desired 7-benzyl thioamidate (9), and the resulting mixture was more readily separated by chromatography than the corresponding mixture of thioamides 6 and 8. Hydrolysis of 9 with HCl afforded 7,8secocyclosporin 11, which was then subjected to Edman degradation conditions to first prepare thiourea 12 and then decapeptide 13. 16 Installation of differentially protected amino acids afforded undecapeptides 14-19. ...
... 15,16 A mixture of 6 and 8 was then treated with benzyl bromide and DBU to give the corresponding benzyl thioamidates; 16 the 4,7-bisbenzyl thioamidate (10) was considerably less polar than the desired 7-benzyl thioamidate (9), and the resulting mixture was more readily separated by chromatography than the corresponding mixture of thioamides 6 and 8. Hydrolysis of 9 with HCl afforded 7,8secocyclosporin 11, which was then subjected to Edman degradation conditions to first prepare thiourea 12 and then decapeptide 13. 16 Installation of differentially protected amino acids afforded undecapeptides 14-19. 14 Treatment with base simultaneously hydrolyzed the acetate at residue 1, the thioester at residue 7, and the Fmoc group of the nascent residue at position 8 to give 7,8-secocyclosporins 20-25. 14 Cyclization with n-propylphosphonic anhydride under dilute conditions gave cyclic undecapeptides 26-31. ...
... 14 Treatment with base simultaneously hydrolyzed the acetate at residue 1, the thioester at residue 7, and the Fmoc group of the nascent residue at position 8 to give 7,8-secocyclosporins 20-25. 14 Cyclization with n-propylphosphonic anhydride under dilute conditions gave cyclic undecapeptides 26-31. Boc-protected amines 26-29 were then treated with CF 3 CO 2 H to afford primary amines 32-35, 14 including [D-Lys] 8 CsA (35), which was ultimately prepared in 6.3% overall yield in 10 steps, which compares favorably to previously reported syntheses. ...
An efficient synthesis of [D-lysine](8)cyclosporin A has been developed. Several analogs of [D-lysine](8)cyclosporin A have been synthesized and show promising anti-HCV activity, particularly compounds 39 and 43, which each exhibit an anti-HCV EC(50)<200 nM, and are each ≥50-fold less immunosuppressive than cyclosporin A.
... Using more extensive chromatography protocols, other minor compounds, Cs E-I (55-59) from the culture of To. inflatum GAMS were also isolated [37]. Traber et al. [38] successfully isolated other minor components, Cs K-Z (61)(62)(63)(64)(65)(66)(67)(68)(69)(70)(71)(72)(73)(74)(75)(76). A summary of the isolations of Cs A-Z (51-76) from the culture of To. inflatum GAMS can be seen in Scheme 4.9. ...
... But, it was found that the fragment condensation strategy was not the best method as it gave a low product yield. Colucci et al. [65] reported a study using BOP-Cl. They found that this coupling agent was good for the synthesis of CsA hexapeptide (2-7) with yields between 78% and 92%. ...
Marine microorganisms and fungi are sources of cyclopeptides and cyclodepsipeptides that have numerous biological activities including antifungal, immunosuppressive, and anticancer. Some have been through clinical trials including didemnin B discussed in this review. These interesting properties of cyclopeptides and cyclodepsipeptides have resulted in a huge increase in research activity in the last few years. In this review, discussion of four families and some miscellaneous cyclopeptides and cyclodepsipeptides is presented.
... Methods used in solution mainly for the preparation of cyclosporine and its analogs are based on the use of the pivaloyl chloride-mixed carbonic anhydride approach, group. [117][118][119][120][121][122] Van der Auwera et al. [123,124] also used BOP-Cl for coupling imino acid residues. BOP-Cl proved more beneficial than the mixed pivalic anhydrides for the preparation of NMAA-rich peptides. ...
Active pharmaceutical ingredients (APIs) can be divided into two types, namely chemical and biological entities. Traditionally, the former has been associated with the so-called small molecules. The revival of peptides in pharmaceutical industry results from their importance in many biological roles. However, low metabolic stability and the lack of oral availability of most peptides is the main drawback for peptide to fulfill that paradigmatic situation. In this regard, efforts are being channeled into addressing this issue by introducing restrictions into the flexible peptide backbone, mainly through N-methyl amino acids (NMAAs) or development of small cyclic peptides. In many cases, both the above restrictions are combined with the aim to enhance oral availability. The synthesis of NMAAs is complex and their introduction into the peptide chain brings additional synthetic challenges and also sometimes leads to side-reactions. Here we discuss the most efficient methods for the synthesis of NMAAs (either in solution or in solid phase) and also their introduction into peptide sequences. Special attention is also given to the detection of side reactions and the most efficient way to prevent them.
... Certain β-peptides have antibacterial , antiproliferative, or hemolytic properties (Chen and Wu 2005b). Mori and Otaka (1994); Vanmiddlesworth et al. (1992) Sphingofungin F (antifungal) Kobayashi et al. (1997) Precursor of β-lactams for synthesis of carbacephems (class of antibiotics) Crocker and Miller (1995) Ebelactone A and B (β-lactone enzyme inhibitor) Paterson and Hulme (1995) Bicycloheptenones Marotta et al. (1994b) Cyclosporine A derivatives (immunosuppressive) Aebi et al. (1990); Colucci et al. (1990); Lynch et al. (1987); Rich et al. (1989); Schmidt and Siegel (1987); Schreiber et al. (1988) (S)-citronellol Hirama et al. (1985) (R)-3-hydroxyheptanoate Anachelin (siderophore of Anabaena cylindrica) Ito et al. (2004) Pravastatin (atherosclerosis/hypercholesteremia agent) Keri et al. (2007) (R)-3-hydroxyhexanoate Analogs of laulimalide (paclitaxel like antimicrotubule agent) Faveau et al. (2006) Appl Microbiol Biotechnol (2010) 87:41–52 ...
The growing awareness of the importance of chirality in conjunction with biological activity has led to an increasing demand for efficient methods for the industrial synthesis of enantiomerically pure compounds. Polyhydroxyalkanotes (PHAs) are a family of polyesters consisting of over 140 chiral R-hydroxycarboxylic acids (R-HAs), representing a promising source for obtaining chiral chemicals from renewable carbon sources. Although some R-HAs have been produced for some time and certain knowledge of the production processes has been gained, large-scale production has not yet been possible. In this article, through analysis of the current advances in production of these acids, we present guidelines for future developments in biotechnological processes for R-HA production.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-010-2530-6) contains supplementary material, which is available to authorized users.
... Their assembly offers wide variety of challenges to synthetic peptide chemists [2]. Though biosynthetic route is the most viable way for the synthesis of Cy [3][4][5][6][7], their chemical synthesis is not very familiar [8][9][10][11][12][13][14][15]. The difficulties encountered during the synthesis of peptides containing N-methylated amino acids are due to their severe steric hindrance offered by them during the coupling. ...
Cyclosporin O (CyO), an immunosuppressent cyclic undecapeptide, was synthesized by convergent approach employing Bsmoc-Nmethyl amino acid fluorides and Potassium Salt of 7-Aza-1-hydroxybenzotriazole (KOAt) in solution by stepwise assembly. The couplings were found to be epimerisation free. The difficulty in the coupling of four consecutive N-methyl amino acids at position 8, 9, 10 and 11 was overcome by repeating the coupling thrice at these critical positions. All the ten protected peptide fragments of CyO starting from the dipeptide to the undecapeptide and final protected as well as CyO were isolated and fully characterized.
... Synthesis of [MeBm2t]' has been described previously (21). Its incorporation into position 1 of CsA, to give [MeBm2t]'-CsA, as well as incorporation of NPBoc-Dap and WBoc-Dab into CsA position 8, was carried out by a modification of Wenger's original strategy (22,23) (V. Kishore and D. H. Rich, manuscript in preparation). ...
Calcineurin, a Ca2+/calmodulin-dependent phosphatase, has recently been identified as a common target for cyclophilin A-cyclosporin A and FK506 binding protein 12-FK506 complexes. This study has examined the structure activity relationships of cyclosporin A (CsA) and three functionally distinct analogues, [MeBm2t]1-CsA, D-diaminobutyryl-8-CsA (Dab8-CsA), and D-diaminopropyl-8-CsA (Dap8-CsA). Immunosuppressive potency in T cell activation models, NF kappa B activation, and IL-2 mRNA transcription has been compared with analogue affinity for cyclophilin A and inhibition of calcineurin phosphatase activity. CsA, Dap8-CsA, and Dab8-CsA bind to cyclophilin A with a similar affinity (Ki 4 to 5 nM as measured by inhibition of prolyl cis-trans isomerase activity), however, Dap8-CsA and Dab8-CsA inhibit T cell activation less than CsA. Although [MeBm2t]-CsA has weak affinity for cyclophilin A (Ki 540 nM), its immunosuppressive potency is similar to that of CsA. Both cyclophilin A-CsA and cyclophilin A-[MeBm2t]1-CsA complexes inhibit calcineurin phosphatase activity in vitro (Ki 114 and 67 nM, respectively). In Jurkat cells exposed to CsA or the analogues for 2 h, endogenous calcineurin phosphatase activity in cell lysates was inhibited by CsA and [MeBm2t]1-CsA (drug concentrations causing 50% reduction in 32PO4 release of 8 and 55 nM, respectively) in proportion to inhibition of T cell activation, IL-2 mRNA transcription, and NF kappa B activation. Dap8-CsA and Dab8-CsA had a minimal effect on endogenous calcineurin phosphatase activity in Jurkat cell lysates. These findings correlate the functional activity of CsA and structural analogues with calcineurin phosphatase activity and support calcineurin as a target for drug action. The Dap8 and Dab8 modifications of CsA, occurring in residue 8, which is exposed to solvent in the cyclophilin A-CsA complex, appears to significantly alter complex affinity for calcineurin.
The first two chapters dealt with formation of new carbon-carbon bonds by processes in which one carbon acts as the nucleophile and the other as the electrophile. In this chapter, we turn our attention to noncarbon nucleophiles. Nucleophilic substitution at both sp
3 and sp
2 centers is used in a variety of synthetic operations, particularly in the inverconversion of functional groups. The mechanistic aspects of nucleophilic substitutions were considered in Part A, Chapters 5 and 8.
VALSPODAR (2) under bar, a cyclic undecapeptide anticancer drug derived from natural Cyclosporin D <(10)under bar>, was labelled with carbon-14 in a nine step synthesis. The sequence started from (-)-[1-C-14]BABS <(1a)under bar>, a highly versatile two-carbon synthon for a broad spectrum of singly/multiply labelled substance classes, which after conversion to (-)-[1-C-14]DPMGBS <(1b)under bar> and subsequent alkylation with isopropyl iodide gave e.p. N-Boc-S-[1-C-14]valine (7) under bar in 46% yield. Coupling to the respective linear decapeptide P-D(8->6), followed by cyclization and selective oxidation afforded the labelled drug substance in an overall radiochemical yield of 9%.
A simple, efficient and racemization free method for the synthesis of peptides employing Fmoc-amino acid chlorides mediated by HOAtDCHA as a co-coupling agent has been described. This protocol is successfully employed in the synthesis of the pentapeptide H-Pro-Gly-VaI-GIy-VaI-OH (PGVGV), and ß-casomorphin (H-Tyr-Pro-Phe-Pro-Gly-OH) in 85 and 80% yields, respectively.
A series of compounds containing a hydroxyethyl-based dipeptide surrogate have been prepared as probes to evaluate the possibility of ICE being an aspartic protease. The aldehyde t-BocAsp(β-t-butyl)H reacted with the organochromium species derived from phenethyl bromide and CrCl2 to give the expected addition product. Lactonization, reprotection of the amine and oxidation with RuCl3 gave the two protected dipeptide surrogates 7a and 7b. These were incorporated into tetra-, penta- and hexapeptide-like molecules and evaluated as inhibitors of the enzyme. The failure of these compounds to inhibit ICE indicated that this enzyme was very unlikely to be an aspartic protease.
Since its isolation in 1970, and discovery of its potent inhibitory activity on T-cell proliferation, cyclosporin A (CsA) has been shown to play a significant role in diverse fields of biology. Furthermore, chemical modification of CsA has led to analogs with distinct biological activities associated with its protein receptor family, cyclophilins.
This review systematically collates the synthetic chemistry performed at each of the eleven amino acids, and provides examples of the utility of such transformations. The various modifications of CsA are traced from early, modest chemistry performed at the unique Bmt residue, through the remarkable use of a polyanion enolate that can be stereoselectively manipulated, and onto application of more recently developed olefin metathesis chemistry to prepare new CsA derivatives with unexpected biological activity.
The myriad biological activities of CsA and its synthetic derivatives have inspired the development of new approaches to modify the CsA ring. In turn, these new CsA derivatives have served as tools in the discovery of new roles for cyclophilins.
This review provides information on the types of cyclosporin derivatives that are available to the many biologists working in this field, and should be of value to the medicinal chemist trying to discover drugs based on CsA. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.
Copyright © 2015. Published by Elsevier B.V.
A highly efficient asymmetric total synthesis of ent-tetrahydrolipstatin (THL) was accomplished from the known epoxy alkenol. The beta-lactone portion of THL was synthesized by the epoxide opening with cyanide and stereoselective enolate alkylation method. The side chain was introduced by cross metathesis of the alkene part with 1-decene. Coupling with a protected leucine completed the synthesis of ent-tetrahydrolipstatin.
The amide bond formation was developed in the early 20th century, however, acylation of hindered or secondary amines still poses a significant challenge. Recently, novel two-component coupling reaction using old functional groups has been established for construction of tertiary amides. It is likely that careful reconsideration of known reactions might produce new valuable chemistry.
Total synthesis of the originally proposed structure of coibamide A, a highly N- and O-methylated cytotoxic marine cyclodepsipeptide, has been accomplished by using a [(4+1)+3+3]-peptide fragment-coupling strategy and careful examination and optimization of the multiple dense N-methylated amide-bond formations. The synthetic sample of the proposed coibamide A could not match the natural product in both 1H and 13C NMR spectra, but was found to exhibit low micromolar cytotoxicity against the proliferation of three tested cancer cells.
The synthesis of a small series of 2-nitroimidazoles in which the β-amino alcohol side chain was amidated with a range of alkylating/acylating functionality is described. Synthetic methodologies were developed that generally provided for selective N-acyl versus N,O-bisacyl products. In vitro, target analogs showed minimal radiosensitization activity, with only a few exhibiting a sensitizer enhancement ratio (SER) >2.0 and C1.6 values comparable to reference agents RB-6145 and RSU-1069. In an assay to determine potential to alkylate biomolecules, representative analogs showed <1% of the alkylating activity of RSU-1069. In vivo, one analog showed an enhancement ratio of 1.6 relative to vehicle control when tested in B6C3F1 mice with an implanted KHT sarcoma. The data reinforce prior findings that there is a correlation between alkylation potential and in vivo activity.
A convergent enantioselective synthesis of panclicin-D has been reported from simple octanal using syn aldol reaction via intramolecular S(N)2 displacement reaction for the first time towards the construction of anti-beta-lactones in panclicin-D. The key steps involved are C-allylation, asymmetric aldolization under Crimmins condition, intramolecular S(N)2 displacement, and Mitsunobu esterification reaction. (C) 2014 Published by Elsevier Ltd.
Activation of Boc-amino acids and Boc-N-methyl amino acids leads to the corresponding NCA. This side reaction explains the low yields obtained when coupling N-methyl amino acids. It was not observed with the Z-or Fmoc-protective groups.
Attempts toward the asymmetric synthesis of (−)-tetrahydrolipstatin are described. A palladium catalyzed Wacker-type reaction to convert an alkene to a ketone, highly diastereoselective reduction of a β-hydroxy ketone, selective oxidation of a diol, and modular synthesis are the key features of the successful approach.
Comparison of different coupling reagents for effective coupling of N-methylated, sterically hindered amino acids under solid-phase peptide synthesis (SPPS) conditions is described. Superior results were obtained with the coupling additive 1-hydroxy-7-azabenzotriazole (HOAt), as well as its uronium salt derivative (HATU), which both produced quantitative couplings. Application of these reagents to the synthesis of the 2–7 sequence found in cyclosporin is reported.
The main product of N-methylated amino acid coupling, when oxybenzotriazole is present in the reagent (BOP, PyBOP, HBPyU) or as an additive (DCC/HOBt), is the weakly reactive benzotriazolyl ester. On the other hand, the corresponding new halogenated reagents PyBroP, PyCloP and PyClU give very good results and can be recommended.
The absolute stereostructure of two N-methylcysteines in kendarimideA (1), a novel linear peptide reversing multidrug resistance (MDR) mediated by P-glycoprotein, was determined by comparison with synthetic model compounds. For this purpose, four diastereomers (17a-d) of the C-terminal tetrapeptides in kendarimide A (1), containing l,l-, l,d-, d,l-, d,d-N-methylcysteinel-N-methylcysteine, were synthesized. The absolute configuration of both the two adjacent N-methylcysteines in 1, which form an eight-membered disulfide ring (ox-[MeCys-McCys]), was elucidated to be l-configuration by comparison of the NMR spectral data of the four model tetrapeptides with that of kendarimide A (1).
An asymmetric synthesis of (-)-tetrahydrolipstatin is described. A palladium-catalyzed regioselective oxidation of ail alkene to a ketone, highly diastereoselective reduction of a beta-hydroxy ketone, selective oxidation of a diol, and modular synthesis are the key features of the synthesis.
L-2,3-trans-3,4-cis-N-Fluorenylmethoxycarbonyl-3,4-dihydroxy-3,4-O-isopropylidineproline (9) has been prepared from D-gulonolactone in nine steps and an overall yield of 22%. Compound 9 has been converted to its allyl ester 13. Compounds 9 and 13 were investigated as building blocks for the incorporation of dihydroxyproline into peptides, with compound 9 serving as a carboxyl component and compound 13 as a precursor to an amino component for peptide coupling reactions. Their utility was demonstrated by the synthesis of dipeptides 11 and 15.
Herein, we describe in full our investigations into the synthesis of grassypeptolide A (1) in 17 linear steps with an overall yield of 11.3 %. In particular, this work features the late-stage introduction of sensitive bis(thiazoline) heterocycles and 31-membered macrocyclization conducted at the sterically congested secondary amide site in superb conversion (72 % yield). Biological evaluation indicated that grassypeptolide A significantly inhibited cancer cell proliferation in a dose-dependent manner. It induced cancer cell apoptosis, which was associated with increased cleavage of poly(ADP-ribose) polymerase (PARP) and decreased expression of bcl-2 and bcl-xL. Furthermore, grassypeptolide A also caused cell cycle redistribution by increasing cells in the G1 phase and decreasing cells in the S and G2 phases. In addition, cell cycle arrest was correlated with downregulation of cyclin D and upregulation of p27 and p21.
Cyclophilins are a family of proteins that bind cyclosporin A (CsA) and possess peptidyl-prolyl cis-trans isomerase activity.
In addition, they are secreted by activated cells and act in a cytokine-like manner, presumably via signaling through a cell
surface cyclophilin receptor. More recently, host-derived cyclophilin A (CyPA) has been shown to be incorporated into HIV-1
virions and its incorporation essential for viral infectivity. Here we present evidence supporting a role for viral-associated
CyPA in the early events of HIV-1 infection. We report that HIV-1 infection of primary peripheral blood mononuclear cells
can be inhibited by: (i) an excess of exogenously added CyPA; (ii) a CsA analogue unable to enter the cells; (iii) neutralizing antibodies to CyPA. Taken together with our observations that recombinant CyPA-induced mobilization of calcium
in immortalized, as well as primary, CD4+ T lymphocytes, and that incubation of T cells with iodinated CyPA, followed by chemical cross-linking, resulted in the formation
of a high molecular mass complex on the cell surface, these results suggest that HIV-1-associated CyPA mediates an early event
in viral infection via interaction with a cellular receptor. This interaction may present a target for anti-HIV therapies
and vaccines.
An efficient synthesis of the cyclic hexdepsipeptide destruxin B from its component residues is described that involves a [3+3] fragment coupling followed by cyclization via the azide method. A novel feature of the synthesis is the use of the Boc-hydrazide protecting group for the C-terminal N-methylalanine residue. This group serves both to inhibit facile diketopiperazine formation from the N-methylvalyl-N-methylalanine dipeptide and as a latent activating group for hexadepsipeptide cyclization.
Convergent solid phase peptide synthesis is a straightforward approach to the synthesis of protein domains with repetitive structures. The peptide H-(Val-His-Leu-Pro-Pro-Pro)(8)-OH corresponding to the N-terminal domain of gamma-zein has been synthesized using the protected segment Fmoc-(Val-His(Trt)-Leu-Pro-Pro-Pro)-OH as synthetic precursor. Solid phase repetitive coupling of the segment was achieved effectively using a synthetic protocol centered on the use of the new 7-aza-1-hydroxybenzotriazole-based reagents HATU and HOAt. The protected peptide segment Fmoc-Val-His(Trt)-Leu-Pro-Pro-Pro-OH was synthesized on a solid-phase using a combination of the base-labile Fmoc group for the alpha-amino protection, the acid-labile trityl group for the protection of the side-chain of histidine, and the highly acid-labile 4-(4-(hydroxymethyl)-3-methoxyphenoxy)butyric acid (HMPB) as handle, which allows the cleavage of the protected segment from the resin by treatment with 1% TFA in CH2Cl2 with complete retention of the amino acid side-chain protecting groups. This approach also allows the same peptide sequence at different levels of protection to be obtained by varying the cleavage program used and demonstrates that the versatile HMPB handle is completely compatible with the use of the trityl group for protection of the side-chain of histidine. The use of a small quantity of pyridine in the purification of this protected peptide by RPMPLC does not affect the Fmoc group and avoids premature imidazole detritylation. Finally, the general synthetic strategy described in this paper avoids the severe problems previously reported on the synthesis of these highly repetitive structures as demonstrated by a careful analysis of the target compounds by cation-exchange HPLC and electrospray MS.
PyBroP (1) and PyCloP (2), two halotripyrrolidinophosphonium hexafluorophosphates, are peptide-coupling reagents highly efficient for coupling N-methylated amino esters, in contrast with PyBOP (3), the hydroxybenzotriazolyl analogue. These halogenophosphonium salts 1 and 2 are convenient (one-pot reactions) stable solids soluble in conventional solvents. Use of them gave an excellent peptide yield with essentially no epimerization. Activation with these reagents probably involves the formation of an (acyloxy)phosphonium, as shown in the case of 2,4,6-trimethylbenzoic acid activation. In the case of reagents 1 and 2, oxazolone and/or a symmetrical anhydride were intermediates which were rapidly aminolyzed. In contrast, the benzotriazolyl ester intermediate which was formed with PyBOP (3) was poorly reactive with N-methylated amino esters. PyBroP (1) and PyCloP (2) were less efficient in the coupling of some Boc-amino acids because of N-carboxyanhydride formation; this was particularly the case when Boc-Val-OH or Boc-MeVal-OH was coupled with MeVal-OMe.
We describe the design and evaluation of structural mimics of tendamistat, a 74-residue proteinaceous inhibitor of alpha-amylase. Cyclic hexapeptides were designed in which the sequence Trp-Arg-Tyr is constrained to the i + 1 to i + 3 positions of a type I beta-turn; these compounds inhibit alpha-amylase with K-i values of 14-32 mu M, significantly more tightly than related linear tri- and hexapeptides. Incorporation of the bicyclic Nagai-Sato type II beta-turn mimic opposite the Trp-Arg-Tyr sequence in a chimeric molecule leads to a weaker inhibitor. NMR studies indicate that the desired beta-turn conformation is adopted by the cyclic hexapeptides but not by the chimeric molecule, supporting the interpretation that the former are indeed acting as small molecule mimics of tendamistat.
Syntheses of cyclosporine analogues are reported wherein the peptide couplings were achieved in solid phase. The Wang resin was used as the solid support, and the peptide couplings commenced with the residue 11 of the cyclosporine skeleton. The couplings proceeded in a stepwide manner up to the residue MeBmt1, using symmetric anhydrides. The peptides were then cleaved off the resin, and the cyclization was achieved in solution using Castro's reagent. The solid-phase synthesis described herein offers a very efficient method for the rapid synthesis of structurally diverse cyclosporine analogues in small quantities. The biological activities of the synthetic cyclosporine analogues were evaluated in two in vitro assays, including the IL-2 reporter gene assay and the cyclophilin binding assay. The structure-activity relationship is discussed.
Twelve selected carboxyl activating reagents have been tested in the β-carbon homologation of D-serine. FDPP and BOP-Cl were the most successful reagents.
The chiral bromo[13,14Cn]acetyl sultams (+)- and (−)-[13,14Cn]BABS 1a, 1b have been demonstrated to be highly efficient, versatile and practical synthons to numerous enantiomerically pure singly and multiply labelled building blocks. The trichlorotitanium enolates derived from 1a, 1b undergo aldol addition reactions with aldehydes providing easily purified, crystalline syn-2-bromo-3-hydroxy [13,14Cn]carboxylic acid derivatives with excellent diastereo-selectivity. These can serve as starting materials for e.p. singly/multiply labelled α-substituted β-hydroxy acids, β-substituted/branched α-hydroxy acids and α-unsubstituted β-hydroxy acids. Furthermore, 1a, 1b can be easily converted to the (+)/(−)-[13,14Cn]DPMGBS 6, (+)/(−)-[13,14Cn]ITCABS 8 and (+)/(−)-[13,14Cn]PABS 10 synthons, which significantly enlarges the spectrum of readiliy accessible intermediates. 6 Provides e.p. labelled α-amino acids, 8 can be employed for the preparation of e.p. labelled α-amino-β-hydroxy acids (threonine type). Synthon 10 reacts with aldehydes to chiral E-configured enoyl sultams 11 which serve as starting materials for a broad variety of e.p. singly/multiply labelled α,β-substituted/branched, acyclic and cyclic carboxylic acid derivatives. Finally, aldehydes, generated by reductive cleavage of the auxiliary from the primary α,β-substituted acyl sultams, react with Ph3P=COOR to give γ,δ-substituted α,β,-unsaturated esters, which in turn can be readily converted to highly functionalized e.p. labelled intermediates. This methodology has been extensively exploited for the synthesis of a broad spectrum of carbon-14 labelled drug substances e.g. Taxol, Valsartan, Everolimus, Lipid X, NVP IMM125, SDZ ISQ844, SDZ PRI05 and the cyclosporin derivatives Valspodar, NVP IMM125, NVP NIM811. Copyright © 2002 John Wiley & Sons, Ltd.
The solid phase synthesis of the cyclic depsipeptide antibiotic lysobactin is described. The natural product was synthesized via a linear approach using mostly an Fmoc-strategy solid phase peptide synthesis (SPPS) with a single purification. A lysobactin analog has also been synthesized displaying nanomolar membrane disruption activity not seen with the natural product.
Various β-lactones were prepared from β-hydroxycarboxylic acids by intramolecular dehydration condensation using MNBA, an effective coupling reagent, along with a nucleophilic catalyst. The transition state that provides the desired 4-membered ring model system is disclosed by density functional theory (DFT) calculations, and the structural features of the transition form are discussed. This method was successfully applied to the asymmetric total synthesis of tetrahydrolipstatin (THL), an antiobestic drug used in clinical treatment to inhibit the activity of pancreatic lipase.
A review with 406 refs. The prepns. of the title compds., their coupling mechanisms, their uses in peptide synthesis and in other applications are presented. [on SciFinder(R)]
The asymmetric total synthesis of (-)-saframycin A, a natural antitumor product of the tetrahydroisoquinoline antitumor antibiotics family, has been accomplished by employing L-tyrosine as the starting chiral building block in 24 steps for the longest linear sequence in an overall yield of 9.7%. The key steps in the synthesis involve stereoselective intermolecular and intramolecular Pictet-Spengler reactions, which induced the correct stereochemistry at C-1 and C-11, respectively. The selective protection-deprotection protocol of an amino group in the two-step transformation from intermediate 10 to 12 and a hydroxyl group in the first two steps resulted in both high selectivity and efficiency of the synthetic route.
The first total synthesis of grassypeptolide, an anticancer cyclodepsipeptide isolated from marine cyanobacteria, has been achieved in 17 steps and an overall 11.3% yield.
Recent developments in the use of isonitriles to furnish secondary and tertiary amide bond formations have been applied to a novel total synthesis of the important cyclic polypeptide cyclosporine A. Specifically, the disclosed synthetic route demonstrates the utility of microwave-mediated carboxylic acid isonitrile couplings, thioacid isonitrile couplings at ambient temperature, and isonitrile-mediated couplings of carboxylic acids and thioacids with amines to form challenging amide bonds.
A convenient synthesis and the biological properties of new amides, esters and other derivatives of trans-stilbene are described. The key synthetic strategies involve the Wittig-Horner reaction of a phosphonium salt 9 and an aldehyde 10 to generate (E)- or (Z)-olefins and a coupling reaction of an acid 12 and various amines 13a-n to give trans-stilbene derivatives 15a-n in high yields. A amide derivative 15g showed three times more in vitro free radical-scavenging activity than resveratrol, while another 15d exhibited strong inhibitory activity against lipopolysaccharide (LPS)(a)-induced NO generation. Allylamide analogue 15a showed the most potent neuroprotective activity in glutamate-induced primary cortical neuron cells.
Amide bond formation is a fundamentally important reaction in organic synthesis, and is typically mediated by one of a myriad of so-called coupling reagents. This critical review is focussed on the most recently developed coupling reagents with particular attention paid to the pros and cons of the plethora of "acronym" based reagents. It aims to demystify the process allowing the chemist to make a sensible and educated choice when carrying out an amide coupling reaction (179 references).
The equilibrium constant for the binding of a spectroscopically invisible ligand to its protein receptor can be determined in a competition experiment, by using a structural analog that contains a reporter group (fluorophor). A novel mathematical treatment of the multiple equilibria allows the analysis to be performed under tight-binding conditions. The equilibrium equation for mixtures of two mutually competitive tight-binding ligands can be expressed in a recursive form, a form in which the dependent variable appears on both sides and the solution is found iteratively. The algorithm is also applicable to the special case of weak binding, where the concentration of the bound ligand can be neglected in the mass balance. The fluorescence displacement method is demonstrated on the determination cyclophilin binding to cyclosporin A (CsA), in competition with its fluorescent derivative, [D-Lys(Dns)]8-CsA.
A novel family of cyclosporin A (CsA) binding proteins was identified by using the biologically active, radioiodinated photoaffinity probe [D-Lys-N epsilon-(4-azido-3-[125I]iodophenyl)propionyl)]8-CsA. In addition to cyclophilin, proteins with molecular masses of 43 kDa and approximately 50-55 kDa were labeled in Jurkat extracts and bovine calf thymus. Sequence analysis of the 43-kDa protein purified from calf thymus and subsequent Western analysis of CsA affinity-purified material from Jurkat extracts identified the 43-kDa component as actin. [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA, a fluorescent analogue of CsA, was prepared and used to measure the binding constants of cyclosporin derivatives to actin by means of a new fluorescence displacement assay. [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA and [N delta-t-butoxycarbonyl diaminobutyryl)]8-CsA bind to bovine actin at physiologically relevant concentrations, with dissociation constants of 60 +/- 33 and 570 +/- 380 nM, respectively. Because the ATPase fragment of heat shock cognate 70 (HSC 70) is structurally related to actin, the yeast homologue SSA1 was tested and found to be radiolabeled by the cyclosporin A photoaffinity reagent. The binding constant for [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA to SSA1 was determined and is 53 +/- 48 nM. These results indicate that actin and the 70-kDa heat shock protein family contain a structurally related domain for binding of cyclosporin A-related peptides.
Four analogs of cyclosporin A (CsA) were synthesized to determine if the biological activities of CsA analogs generated by multiple amino acid replacements are predictable from the effects on biological activity of analogs with single residue changes. CsA analogs [Phe7]CsA (8a), [D-MeAla3,Phe7]CsA (8b), [D-Ser8,Phe7]CsA (8c), and [D-MeAla3,Phe7,D-Ser8]CsA (8d) were designed by modification of positions 3, 7, and 8, which are adjacent to one effector region of the cyclophilin-bound CsA complex. The syntheses of CsA analogs 8a-d were carried out by suitable modifications of the reported strategy. Each analog was characterized by NMR in deuterated chloroform and DMSO solutions, and their biological activities as inhibitors of cis-trans-peptidyl prolyl isomerase (PPIase), inhibitors of proliferation in BDF1 mouse spleen cells stimulated with concanavalin A (Con A), and inhibitors of IL-2 release stimulated with PMA/ionomycin by Jurkat cells were determined. Incorporation of the phenylalanine residue in position 7 diminished activities 5-8-fold. Substitution at position 3 decreased activity nearly 2-fold, and substitution at position 8 did not lower activities. However, when all three modifications (D-MeAla3,Phe7, and D-Ser8) were incorporated into one molecule, the resulting analog, 8d, was found to bind more tightly to cyclophilin than CsA (Ki = 3 +/- 1.5 vs 6 +/- 2 nM) and to produce the full immunosuppressive effect in the other assay systems. Our structure-activity results show that combinations of substitutions that individually lower PPIase or immunosuppressive activity produce fully active analogs when combined in a single compound. These results suggest that other, multimodified CsA derivatives may be discovered that possess excellent or improved immunosuppressive activities even though they contain a substitution that otherwise reduces immunosuppressive activity.
Coupling of Fmoc-amino acid chlorides can be mediated by the potassium salt of 1-hydroxybenzotriazole (KOBt), the reaction being carried out in an organic medium. The use of a base like NaHCO3/Na2CO3 or DIEA/NMM/pyridine is not necessary. Coupling is fast and racemization free; the work-up, isolation of the product and scale-up are easy. The pentapeptide sequence of Fmoc-[Leu]enkephalin was thus synthesized in the solution phase on a 5 mmol scale without isolation of any intermediate. Acylation of C-protected N-methylamino acid esters by Fmoc-N-methylamino acid chlorides by this procedure is also feasible, as demonstrated by the synthesis of cyclosporin A fragments 4-7 and 8-11. The peptides obtained in high yields were crystalline solids, unlike earlier reports in which they were obtained mostly as oily or foamy intermediates. They showed spectral properties identical with those of the authentic compounds.
N-tert-Butoxycarbonylamino acids (Boc-Xaa-OH) were coupled with p-nitrophenol (HONp) in dichloromethane using N,N'-dicyclohexylcarbodiimide (DCC) and N-ethyl-N'(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and the products were identified and quantitated by high-performance liquid chromatography. Boc-Xaa-OH with Xaa = Val was coupled also with pentafluorophenol (HOPf) and hydroxy-containing additives (HOR), and the products were similarly determined as the methylamides. EDC-mediated reactions of Boc-Xaa-OH gave 8-25% of Boc-Xaa-Xaa-OR as well as Boc-Xaa-OR for R = Ph, Np, Pf, benzotriazole (Bt) and 5-norbornene-endo-2,3-dicarboxamide; DCC-mediated couplings, 5-7% for R = Np and Bt. No dimer was formed in couplings with N-hydroxysuccinimide or 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine. Dimerization was eliminated from DCC-mediated reactions by the addition of 1 equiv. of N-methylmorpholine, from the EDC-mediated reactions by carrying them out in pyridine. Dimerization is attributed to formation of the intermediate 2-alkoxy-5(4H)-oxazolone that undergoes fragmentation to the N-carboxyanhydride, which reacts with the alcohol giving amino acid ester. Ester produces dimer by aminolysis of the O-acylisourea. Decomposition (1.4%) was also detected by analysis for H-Val-Phe-OMe in DCC-mediated reactions of Boc-valine with H-Phe-OMe, and was demonstrated to be caused by the hydrochloride of the ester salt that had been neutralized with N-methylmorpholine. Decomposition was eliminated by the addition of 5% of pyridine, which also had the beneficial effect of suppressing N-acylurea formation.
A hydroxide-mediated Fmoc/methyl ester double deprotection procedure followed by an improved bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl) cyclisation reaction are reported. This novel methodology was applied to the synthesis of three new HC-toxin analogues.
Allyl(All) and Allyloxycarbonyl(Alloc) amino-acid derivatives are deprotected through palladium-catalyzed hydrostannolysis by Bu3SnH in a highly selective manner. Benzyl and benzyloxycarbonyl groups are stable under these conditions. Moreover the allyl and Alloc groups seem orthogonal to the t-butyl and t-butoxycarbonyl protecting groups.
A novel method for the cleavage of the Aloc protecting group (Aloc = allyloxycarbonyl) permits the effective and selective synthesis of sensitive peptide derivatives, e.g. with serine residues. The protecting group can be removed from peptide esters such as Aloc‐Phe‐Ser( t Bu)‐O t Bu via catalytic allyl transfer with [Pd(PPh 3 ) 4 ] to dimendone; the t Bu groups are retained. In contrast, with CF 3 COOH only the t Bu groups are removed from the Aloc‐ t Bu esters.
With the bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl) coupling reagent, synthesis of the cyclosporine A 8-11 tetrapeptide has been carried out utilizing both Fmoc- and Cbz-N-protection of the intermediates. The former protecting group allows a very time efficient sequence where isolation of the free imino fragment is obviated and demonstrates the potential for applications to solid phase. Cbz protection is more economical for larger scale synthesis, however, and provides more stable intermediates. In both cases remarkable chemical efficiency was achieved, the fully protected tetrapeptide being obtained in 66% and 73% yields, respectively, from the constituent protected amino acids. By means of the Fmoc procedure, diastereomers at each stage of coupling have been separately prepared. HPLC analysis, using these compounds as internal standards, has shown that racemization in all cases was less than 1%.
Les analogues sont prepares par cyclisation d'undecapeptides; l'aminoacide en 1 est remplace par la N-methyl threonine, l'acide methylamino-2 butyrique (A) ou l'hydroxy-3 N-methyl leucine. Synthese egalement de A-1 sarcosyl-10 cyclosporine A. Activites biologiques
The chiral glycine synthon 3c, as its derived stannous enolate, has been demonstrated to undergo a highly syn diastereoselective aldol addition reaction with representative aldehydes to give the adducts 5 (R = C6H5, Me, Me2CH) in yields ranging from 71 to 92%. The utility of these intermediates has been demonstrated via the subsequent three-step transformation of these adducts to the enantiomerically pure N-methyl β-hydroxy amino acids 1. This reaction methodology has been applied to the asymmetric synthesis of (4R)-4-((E)-2-butenyl)-4,N-dimethyl-L-threonine (1a), an important constituent in the immunosuppressant peptide cyclosporine. Several additional structural analogues of 1a were also prepared in conjunction with this study.
Sharpless epoxidation of the readily available dienol , followed by regiospecific opening with Me2CuLi/BF3·Et2O yields, after protecting group manipulation, the monoprotected diol . Swern oxidation gives the cyclic carbaminol which is easily manipulated to α-cyano oxazolidinone and thus, by literature methods, to the title compound. Using this procedure, good overall yield and high enantiomeric purity are obtained.
The heptapeptide H-MeBmt-Abu-Sar-MeLeu-Val-MeLeu-Ala-OBzl (20) was synthesized for coupling with the previously described cyclosporine tetrapeptide sequence Boc-D-Ala-MeLeu-MeVal-OH (21). The product of the coupling, the undecapeptide Boc-D-Ala-MeLeu-MeLeu-MeVal-MeBmt-abu-Sar-MeLeu-Val-MeLeu-Ala-OBzl (22), was then deprotected and cyclized to cyclosporine (1).
The tetrapeptide diastereoisomer Boc-D-ala-MeLeu-MeLeu-D-MeVAl-OH (23) could also be used as a starting material to produce selectively the desired undecapeptide 22. In this case, the N-methyl-D-valine unit, was selectively isomerized to the L-from by using the appropriate condensing agent. The diastereoisomeric undecapeptide Boc-D-ala-MeLeu-MeLeu-D-MeVal-MeBmt-Abu-Sar-MeLeu-Val-MeLeuAla-OBzl (24) was also synthesized starting from 21 by using the mixed pivalic anhydride method to selectively invert the configuration of the N-methyl-L-valine. The structure of the undecapeptide 24 was confirmed by deprotection and cyclization to ‘cyclosporin H’, a natural product known to have the structure [D-MeVal11]cyclosporine (2).
Cyclosporin A has been shown to possess antimalarial activity in mice infected withPlasmodium berghei NK 65 orPlasmodium chabaudi. Significant antimalarial effects were obtained with five daily oral doses of 25 mg/kg. Cyclosporin A treatment started concurrently with the inoculation of parasites was less effective than treatment started when parasitaemia was already established. Evidence so far suggests that the antimalarial action results from a direct toxic effect on the parasite. Combined treatment with Cyclosporin A and Pyrimethamine indicated that the two compounds may act synergistically.
The enzyme peptidyl-prolyl cis-trans isomerase (PPIase) was recently discovered in mammalian tissues and purified from porcine kidney. It catalyses the slow cis-trans isomerization of proline peptide (Xaa-Pro) bonds in oligopeptides and accelerates slow, rate-limiting steps in the folding of several proteins. Here, we report the N-terminal sequence of PPIase together with further chemical and enzymatic properties. The results indicate that this enzyme is probably identical to cyclophilin, a recently discovered mammalian protein which binds tightly to cyclosporin A (CsA). Cyclophilin is thought to be linked to the immunosuppressive action of CsA. The first 38 amino-acid residues of porcine PPIase and of bovine cyclophilin are identical and the two proteins both have a relative molecular mass of about 17,000 (ref. 7). The catalysis of prolyl isomerization in oligopeptides and of protein folding by PPIase are strongly inhibited in the presence of low levels of CsA. The activities of both PPIase and cyclophilin depend on a single sulphydryl group. At present it is unknown whether the inhibition of prolyl isomerase activity is related with the immunosuppressive action of CsA.
Peptidyl-prolyl cis-trans isomerase (PPIase) catalyses the cis-trans isomerization of proline imidic peptide bonds in oligopeptides and has been shown to accelerate the refolding of several proteins in vitro. Its activity has been detected in yeast, insects and Escherichia coli as well as in mammals, and it is though to be essential for protein folding during protein synthesis in the cell. We purified PPIase from pig kidney and found that its amino-acid sequence is identical to that reported for bovine cyclophilin, a protein known to bind the immunosuppressive drug, cyclosporin A (ref. 5). To investigate the functional relationship between PPIase and cyclophilin we examined the effect of cyclosporin A on PPIase activity and found that it was inhibitory. Thus we propose that the peptidyl-prolyl cis-trans isomerizing activity of PPIase may be involved in events, such as those occurring early in T-cell activation, that are suppressed by cyclosporin A.
Administration of cyclosporin A, a new selective immunosuppressive agent, to mice infected with Schistosoma mansoni resulted in a significant reduction in the number of mature and immature male and, to a greater extent, female worms. With lower, subeffective, doses a reduction in hemoglobinase activity and protein content of female schistosomes is produced. Evidence available so far suggests that the antischistosomal effects of cyclosporin A are mediated through a stimulation of host mechanisms directed against the parasite.