SYNTHESIS OF TWO ANALOGS OF A CYCLIC HEXAPEPTIDE WITH DISULFIDE BRIDGE CORRESPONDING TO BOVINE PROTHROMBIN PRECURSOR SEQUENCE 18–23: Extent of Carboxylation by Vitamin K-Dependent Carboxylase

The synthesis of two analogs of sequence 18–23 of bovine prothrombin precursor is described. Hexapeptides Boc-Cys(Acm)-Leu-Glu(OBzl)-Glu (OBzl)-Pro-Cys(Acm)-OBzl and Ac-Cys(Acm)-Leu-Glu(OBzl)-Glu(OBzl)-Pro-Cys(Acm)-OMe were synthesized in solution by stepwise addition of Boc-amino acids using dicyclohexylcarbodiimide/N-hydroxybenzotriazole as the coupling reagent. The acetamidomethyl groups were cleaved and oxidized, using iodine in methanol, to the protected cyclic disulfide in 62–69% yield. The 0-benzyl groups were removed either by treatment with anhydrous hydrogen fluoride or hydrogen bromide in trifluoroacetic acid to give the cyclic hexapeptide disulfides, R₁-Cys-Leu-Glu-Glu-Pro-Cys-OR₂ where R₁, = H or Ac and R₂ = H or CH₃. The cyclic hexapeptides were evaluated as substrates for vitamin K-dependent carboxylase. Both peptides are unusually poor substrates for the carboxylase, and each appears to inhibit carboxylation of Phe-Leu-Glu-Glu-Leu, a good substrate for the enzyme.     

Syntheses and properties of tertiary peptide bond-containing polypeptides.

Oligo(Leu)s containing Pro or (Dmob) Leu residues were prepared by the stepwise elongation and fragment condensation methods. The peptides prepared were the following: Boc-Leu_n-OBzl (n = 3–6 and 9), Boc-Pro-Leu₃-OBzl, Boc-Leu_n-Pro-OBzl (n = 3–5), Boc-Leu_n-Pro-Leu₃-OBzl (n = 3 and 4), Boc-(Leu₄-Pro)₂-OBzl, Boc-Leu₃-Pro-Leu_n-Pro-Leu₃-OBzl (n = 6 and 7), Boc-Leu₃-Gly-(Dmob) Leu-OMe, Boc-[Leu₃-Gly-(Dmob) Leu-Leu₃]_n-OBzl (n = 1 and 2). The insertion of the Pro and Gly-(Dmob) Leu residues into a peptide chain caused “the peptide segment separation” and achieved remarkable solubility improvement of N- and C-protected oligo(Leu) derivatives in a variety of organic solvents. The conformations of typical peptides in the solid state and in DMSO were also investigated using i.r. spectroscopy. The incorporation of tertiary peptide bonds in a central position of the β-sheet structure induces the onset of an unordered structure in the solid state. In DMSO, peptides are efficiently subjected to solvation to have the unordered structure.     

Synthesis of O-glycosylated tuftsins by utilizing threonine derivatives containing an unprotected monosaccharide moiety

Synthesis is described of four tuftsin derivatives containing a D-ghcopyranosyl or a D-galactopyranosyl unit covalently linked to the hydroxy side chain function of the threonine residue through either an α or βO-glycosidic linkage. Fmoc-threonine derivatives containing the suitable unprotected sugar were used for incorporating the O-glycosylated amino acid residue. Z-Thr[α-Glc(OBzl)₄]-OBzl and Z-Thr[α-Gal(OBzl)₄]-OBzl were prepared from the tetra-O-benzylated sugar and Z-Thr-OBzl by the trichloroacetimidate method in the presence of trimethylsilyl trifluoromethane sulfonate. The α glycosylated threonine derivatives were converted into Fmoc-Thr(α-G1c)-OH and Fmoc-Thr(α-Gal)-OH by catalytic hydrogenation followed by acylation with Fmoc-OSu. β-Glucosylation and β-galactosylation of threonine were carried out by reacting the proper per-O-acetylated sugar with Z-Thr-OBzl and boron trifluoride ethyl etherate in dichloromethane. Catalytic hydrogenation of the β-O-glycosylated threonine derivatives followed by acylation with Fmoc-OSu and deacetylation with methanolic hydrazine yielded Fmoc-Thr(β-Glc)-OH and Fmoc-Thr(β-Gal)-OH, respectively. The O-glycosylated threonine derivatives were then reacted with H-Lys(Z)-Pro-Arg(NO₂)-OBzl in the presence of DCC and HOBt and the resulting glycosylated tuftsin derivatives were fully deblocked by catalytic hydrogenation, purified by HPLC, and characterized by optical rotation, amino acid analysis, and ¹H NMR. The β-galactosylated tuftsin was also prepared by the continuous flow solid phase procedure.     

Chemical synthesis of phosphorylated peptides of the carboxy-terminal domain of human p53 by a segment condensation method

A segment condensation method was developed for the chemical synthesis of large (>90 amino acid) phosphopeptides and was used to produce phosphorylated and non-phosphorylated derivatives of the C-terminal tetramerization and regulatory domains of human p53 (residues 303-393). Efficient condensation synthesis of the 91 residue p53 domain was achieved in two steps. The non-phosphorylated N-terminal segment p53(303-334) (1) and its derivative phosphorylated at serine 315 (1P315), and the non-phosphorylated middle segment p53(335-360) (2), were synthesized as partially protected peptide thioesters in the solid phase using Boc chemistry. The C-terminal segment p53(361-393) (3) and its derivative phosphorylated at serine 392 (3P392) were synthesized as partially protected peptides in the solid phase using Fmoc chemistry. Phosphoamino acid was incorporated into the N-terminal segment (1P315) at the residue corresponding to p53 serine 315 as Boc-Ser(PO₃(Bzl)₂)-OH during synthesis. Serine 392 in the C-terminal segment was selectively phosphorylated after synthesis by phosphitylation followed by oxidation. A derivative phosphorylated at serine 378 was synthesized in a one-step condensation of the unphosphorylated N-terminal segment (1) and the phosphorylated long C-terminal segment p53(335-393) (2-3P378). Yields of the ligated peptides after removal of the protecting groups and HPLC purification averaged 60% for the first condensation and 35% for the second condensation. All five p53 peptides exhibited monomer-tetramer association as determined by analytical ultracentrifu-gation. Circular dichroism spectroscopy revealed that phosphorylation at Ser315 increased the α-helical content, which was abolished when Ser392 also was phosphorylated, suggesting an interaction between N-terminal and C-terminal residues of the C-terminal domain of p53. © Munksgaard 1996.     

Efficient solution-phase synthesis of multiple O-phosphoseryl-containing peptides related to casein and statherin

The multiple Ser(P)-containing peptides, H-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-NHMe-TFA, H-Asp-Ser(P)-Ser(P)-Glu-Glu-NHMe-TFA and H-Glu-Ser(P)-Ser(^P)-Glu-Glu-NHMe-TFA were prepared by the use of Boc-Ser(PO₃Ph₂)-OH in the Boc mode of solution phase peptide synthesis followed by platinum-mediated hydrogenolytic de-protection of the Ser(PO₃Ph₂)-containing peptides. The protected peptides were assembled using the mixed anhydride coupling methods with 40% TFA/CH₂C1₂ used for removal of the Boc group from intermediate Boc-protected peptides.     

FIBRINOPEPTIDES. III. SYNTHESIS OF THE PROTECTED NONAPEPTIDE SEQUENCE OF FIBRINOPEPTIDE-B OF GREEN MONKEY

The protected nonapeptide, Z-Asn-Glu(OBzl)-Glu(OBzl)-Gly-Leu-Phe-Gly-Gly- Arg(NO₂)OMe, corresponding to the amino acid sequence present in fibrinopeptide-B of green monkey, has been synthesised by conventional methods.     

Synthesis of the C-terminal half of thymosin α1 by the polymeric reagent method

In this report we further show the utility and efficiency of polymer-bound 1-hydroxybenzotriazole (PHBT) as an almost ideal support for the polymeric reagent method of peptide synthesis. This was demonstrated by the synthesis of thymosin α₁ (15–28), in which two suitably blocked segments, Boc-Asp (OtBu)-Leu-Lys (2Cz)-Glu (OBzl)-Lys (2Cz)-Lys (2Cz)-OH ( 3 ) and Boc-Glu (OBzl)-Val-Val-Glu (OBzl)-Glu (OBzl)-Ala-Glu (OBzl)-Asn-OBzl ( 2 ), were prepared entirely by utilizing PHBT activation for each coupling step. After appropriate deblocking of 2 , segments 2 and 3 were coupled by the DCC-HOBT method, followed by complete deblocking and ion-exchange chromatographic purification, affording the C-terminal half of thymosin α₁, H-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH ( 1 ).     

Synthesis and interaction with metal ions of cyclic oligopeptides bearing carboxyl groups

Cyclic di- and tetrapeptides bearing carboxyl or carboxylate groups, cyclo[Glu(OBzl)-Glu(OMe)], cyclo [Glu-Glu(OMe)], cyclo(Glu-Glu), cyclo[Glu(OMe)-Pro)₂ and cyclo(Glu-Pro)₂ were synthesized and investigated on the intramolecular interaction of carboxyl side chains in the complexation with metal ions in relation with the conformation. The three kinds of cyclic dipeptides were found to take a flagpole boat conformation. Folded conformation of side chains was predominant for cyclo[Glu(OBzl)-Glu(OMe)] and cyclo[Glu-Glu(OMe)]. However, cyclo(Glu-Glu) took an unfolded conformation. Intramolecular interaction of carboxyl groups was observed neither in free state nor in complexation with metal ions. The intramolecular interaction of carboxyl groups was observed in the case of cyclo(Glu-Pro)₂ in the absence of metal ions added. Cyclo[Glu(OMe)-Pro], and cyclo(Glu-Pro)₂ formed a complex with Ca²⁺ and Ba²⁺ without participation of side chains.     

Synthesis of potent agonists of Substance P by replacement of Met11 with Glu(OBzl) and N-terminal glutamine with Glp of the C-terminal hexapeptide and heptapeptide of Substance P

The analogues [Glp⁶,Glu(OBzl)¹¹]SP_(6-11) and [Glp⁵,Glu(OBzl)¹¹]SP_(5-11)) of the C-terminal hexapeptide and heptapeptide of Substance P have been synthesized by conventional solution methods. In each analogue the N-terminal glutamine has been replaced by pyroglutamic acid, while the COOCH₂C₆H₅ ester group has replaced the SCH₃ group of the Met¹¹ side chain. The in vitro activity of both analogues has been determined on three biological preparations: guinea pig ileum (GPI), rat vas deferens (RVD) and rat portal vein (RPV). The results showed that both analogues are highly potent and selective agonists on GPI through the NK-1 receptor. They are more potent than SP itself, with 1.54 and 1.25 respective values of relative potency on GPI. Their selectivity has been studied by utilizing atropine-treated guinea pig ileum (GPI + At). The analogues showed low activity on RVD and RPV tissues, which represent NK-2 and NK-3 monoreceptor assay, respectively. © Munksgaard 1995.     

Synthesis,conformation, and biological activity of the carbohydrate-free vespulakinin 1

Synthesis of the carbohydrate-free heptadecapeptide corresponding to the amino acid sequence of vespulakinin 1 was achieved by the continuous flow solid phase procedure on 4-hydroxymethyl-phenoxyacetyl-norleucyl derivatized Kieselguhr-supported polydimethylacrylamide resin, as well as by a combination of solid phase and solution syntheses. Preformed Fmoc-amino acid symmetrical anhydrides (Boc derivative for the N-terminal residue) were used for amine acylation in the continuous flow method. Serine and threonine were side chain protected as tert.-butyl ethers and the 4-methoxy-2, 3, 6,-trimethyl-benzenesulfonyl group was used for masking the guanidino function of arginine residues. After cleavage from the resin the final peptide was purified by ion exchange chromatography and characterized by amino acid analysis, high voltage electrophoresis, and RP-HPLC analysis. Alternatively, the protected N-terminal octapeptide, Fmoc-Thr(Bu^t)-Ala-Thr(Bu^t)-Thr(Bu^t)-Arg(Mtr)-Arg-(Mtr)-Arg(Mtr)-Gly-OH was prepared on 4-hydroxymethyl-3-methoxyphenoxyacetyl-norleucyl derivatized Kieselguhr-supported polydimethylacrylamide resin and the C-terminal nonapeptide H-Arg(NO₂)-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-(NO₂)-OBzl was synthesized in solution through the fragment condensation method. The two fragments were coupled by the DCC-HOBt procedure and the resulting heptadecapeptide was deblocked and purified. The conformational features of the synthesized peptides are reported. Preliminary pharmacological experiments indicated that carbohydrate-free vespulakinin 1 is more potent than bradykinin in lowering rat blood pressure.     

Synthesis and biological activity of Substance P C-terminal hexapeptide and heptapeptide analogues

The analogues [Glu(OBzl)¹¹]SP_6–11 and [Glu(OBzl)¹¹]SP_5–11 of the C-terminal hexapeptide and heptapeptide of Substance P have been synthesized by conventional solution methods. In each analogue the SCH₃ group of Met¹¹ is replaced by the COOCH₂C₆H₅ group. The in vitro activity of both analogues has been determined on three biological preparations: guinea pig ileum (GPI), rat vas deferens (RVD), and rat portal vein (RPV). The selectivity for the different receptors has been studied by utilizing atropine-treated guinea pig ileum (GPI + At). The results showed that both analogues are mainly active on GPI through the NK-1 receptor and that both analogues are equipotent to Substance P.     

Further studies on the use of 2,2,2-trichloroethyl groups for phosphate protection in phosphoserine peptide synthesis

Boc-Ser(PO₃Tc₂)-OH, Z-Ser(PO₃Tc₂)-OH and Fmoc-Ser(PO₃Tc₂)-OH, derivatives useful for peptide synthesis, have been obtained in high yields by acylation of H-Ser(PO₃Tc₂)-OHCF₃COOH. The latter was obtained from Boc- or Z-Ser(PO₃Tc₂)-OBzl by simultaneous removal of the amino- and carboxy-protecting groups by Pd-catalyzed hydrogenolysis in acetic acid-trifiuoroacetic acid solution. Removal of the Tc-protecting group was efficiently achieved by hydrogenolysis in aqueous ethanol.     

Synthesis and solubility properties of peptide fragments of human hemoglobin α-chain (123-136)

The solubility prediction method for protected peptides was successfully applied to relatively small peptide fragments of human hemoglobin α-chain (123-136) which contained various polar amino acid residues such as Asp(OBzl), Glu(OBzl), Lys(Z), Ser(Bzl), and Thr(Bzl). As reported previously for hydrophobic peptides and human proinsulin C-peptide fragments, solubility data indicated that the insolubility of protected peptides having a <P_C > value below 0.90 appeared to begin at the octa- or nonapeptide sequence level and that β-sheet structure played an important role in the insolubility of peptides. When a peptide has a β-sheet structure in the solid state, we can clearly determine the critical chain length for peptide insolubility, the solubility dependence on solvent properties, and the solubility independence of amino acid compositions of peptides.     

Peptide N-alkylamides by solid phase synthesis*

Three new resins have been developed that allow for the solid phase synthesis of C-terminal peptide N-alkylamides using Boc amino acids, usual side chain protecting groups and hydrogen fluoride cleavage and deprotection. These resins were prepared by reacting the appropriate alkylamine (NH₂ CH₃, NH₂ CH₂ CH₃, NH₂ CH₂ CF₃) to Merrifield's 1% divinylbenzene cross-linked chloromethylated polystyrene resin. The application of these resins to the synthesis of C-terminal GnRH N-alkylamides illustrates the versatility of this approach. GnRH analogs were tested for their ability to release LH from cultured rat anterior pituitary cells. [D Glu⁶, Pro⁹-NHCH₂ CH₃]-GnRH was synthesized for the first time using the solid phase approach and found to be three times more potent than [D Glu⁶]-GnRH. Other analogs including [D Trp⁶, Pro⁹-NHCH₂ CH₃]-GnRH, [D Ala⁶, Pro⁹-NHCH₂ CF₃]-GnRH and related peptides were found to be equipotent and to have the same properties (HPLC retention times, amino acid analysis and specific rotation) as the corresponding peptides synthesized using less amenable strategies; yields were equivalent or better than those reported earlier.     

Optimized aminolysis conditions for cleavage of N‐protected hydrophobic peptides from solid‐phase resins

Abstract: Solid‐phase synthesis and aminolysis cleavage conditions were optimized to obtain N‐ and C‐terminally protected hydrophobic peptides with both high quality and yield. Uncharged ‘WALP’ peptides, consisting of a central (Leu‐Ala)_n repeating unit (where n = 5, 10.5 or 11.5) flanked on both sides by Trp ‘anchors’, and gramicidin A (gA) were synthesized using 9‐fluorenylmethoxycarbonyl chemistry from either Wang or Merrifield resins. For WALP peptides, the N‐terminal amino acid was capped by coupling N‐acetyl‐ or N‐formyl‐Ala or ‐Gly to the peptide/resin or by formylation of the completed peptide/resin with para‐nitrophenylformate (p‐NPF). N‐Terminal acetyl‐ or formyl‐Ala racemized when coupled as an HOBt‐ester to the resin‐bound peptide, but not when the peptide was formylated with p‐NPF. Racemization was avoided at the last step by completing the peptide with acetyl‐ or formyl‐Gly. For both WALP peptides and gA, cleavage conditions using ethanolamine or ethylenediamine were optimized as functions of solvent, time, temperature and resin type. For WALP peptides, maximum yields of highly pure peptide were obtained by cleavage with 20% ethanolamine or ethylenediamine in 80% dichloromethane for 48 h at 24°C. N‐Acetyl‐protected WALP peptides consistently gave higher yields than those protected with N‐formyl. For gA, cleavage with 20% ethanolamine or ethylenediamine in 80% dimethylformamide for 48 h at 24°C gave excellent results. For both WALP peptides and gA, decreasing the cleavage time to 4 h and increasing the temperature to 40–55°C resulted in significantly lower yields. The inclusion of hexafluoroisopropanol in the cleavage solvent mixture did not improve yields for either gA or WALP peptides.     

Solid-phase synthesis of protected peptides using new cobalt(III) ammine linkers†

Cobalt(III) ammine complexes of the type cis-[CoL₄(4-AMB)O-AA-Boc](CF₃SO₃)₂, where L₄= bisethylenediamine (en)₂ or tetraammine (NH₃)₄, and 4-AMB = 4-(aminomethyl)benzoic acid, have been synthesized and used as linkers to polystyrene resins for solid-phase synthesis of protected peptides. Boc/t-Bu-protected [Leu⁵]enkephalin was assembled on the two different Co(III) resins, and then cleaved from the resins by reduction of the Co(III) center in 93–96%; yield. HPLC-purified protected [Leu⁵]enkephalin was obtained in 67–69% overall yield and characterized by amino acid analysis and ¹H NMR. Stepwise synthesis on the Co(en)₂-resin was also used in the assembly of Boc-Asp(OcHex)-Arg(Mts)-Gly-Asp(OcHex)-Ala-Pro-Lys(2Cl-Z)-Gly-OH, a sequence from collagen α1 Type 1. The protected peptide was cleaved from the Co(III) resin in 74% yield, and the HPLC-purified nonapeptide was characterized by amino acid analysis, ¹H NMR and liquid secondary-ion mass spectrometry (LSIMS). New routes are described for the synthesis of isomerically pure Co(III) anchor complexes. The Co(III) resins were found to be compatible with both the tert-butyloxycarbonyl (Boc) and the 9-fluorenylmethoxycarbonyl (Fmoc) N^α-protecting group strategies used in solid-phase peptide synthesis.     

Solid-phase synthesis of H- and methylphosphonopeptides

We introduce solid-phase syntheses of H- and methylphosphonopeptides, giving access for the first time to a new class of mimics for o-phosphoamino acids. The model peptides H-GlyGlyXaaAla-OH (Xaa = Ser, Thr) were synthesized on a solid-phase using Fmoc/^tBu strategy and HBTU/HOBt activation by incorporation of hydroxyl-protected serine and threonine. As selectively cleavable hydroxyl-protecting groups we used triphenylmethyl and tert-butyldimethylsilyl for both amino acids, as described in the literature. All peptides were phosphitilated with O,O-di-tert-butyl-N,N-diethylphosphoramidite and yielded H-phosphonopeptides after trifluoroacetic acid cleavage. Alternatively we phosphonylated the peptides with O-tert-butyl-N,N-diethyl-P-methylphosphonamidite, which was synthesized by a two-step one-pot procedure starting from commercially available chemicals. All H- and methylphosphonopeptides were obtained in high purities and yields, as shown by reversed-phase high-performance liquid chromatography and anion-exchange chromatography. The phosphonopeptides were characterized by ¹H and ³¹P NMR. We confirmed their molecular masses by electrospray mass spectrometry and analyzed their fragmentation schemes, which seemed to be characteristic for each class of analogues. The H-phosphonopeptides lost phosphonic acid (H₃PO₃, 82 mass units) and the methylphosphonopeptides lost methylphosphonic acid (MeH₂PO₃, 96 mass units). Both H- and methylphosphonopeptides represent a new and simply accessible class of mimics for phosphopeptides. Compared with the corresponding phosphopeptides all phosphonopeptides were synthesized in higher yields and purities (>80%). © Munksgaard 1996.     

A solid‐phase synthetic strategy for the preparation of peptide‐based affinity labels: synthesis of dynorphin A analogs

Abstract: Solid‐phase synthetic methodology was developed for the preparation of peptide‐based affinity labels. The initial peptides synthesized were dynorphin A (Dyn A) analogs [Phe(p‐X)⁴,d ‐Pro¹⁰]Dyn A(1–11)NH₂ containing isothiocyanate (X = –N=C=S) and bromoacetamide (X = –NHCOCH₂Br) groups. The peptides were assembled on solid supports using Fmoc‐protected amino acids, and the side chain amine to be functionalized, Phe(p‐NH₂), was protected by the Alloc (allyloxycarbonyl) group. Following removal of the Alloc group by palladium(0), the reactive isothiocyanate and bromoacetamide functionalities were successfully introduced while the peptides were still attached to the resin. Synthesis of these peptides was carried out on polystyrene (PS) and polyethylene glycol–polystyrene (PEG–PS) resins containing the PAL [peptide amide linker, 5‐(4‐Fmoc‐aminomethyl‐3,5‐dimethoxyphenoxy)valeric acid] linker. Both the rate of Alloc deprotection and the purity of the crude affinity‐labeled peptides obtained were found to be dependent on the resin used for peptide assembly.     

Approaches to the synthesis of retro-inverso peptides

Partial retro-inverso modification of biologically active peptides is described as a topochemical alteration of the backbone to prevent enzymatic degradation. The preparation of gem-diaminoalkyl residues from peptide amides using the reagent [bis(trifluoroacetoxy)iodo] benzene (TIB) is discussed. Treatment of N-t-butyloxycarbonyl tyrosine and N-t-butyloxycarbonyl tryptophan with this reagent led to decomposition of the protected amino acids. Protecting the tyrosine and tryptophan residues by t-butyl ether and Nⁱⁿ-formyl groups, respectively, prevented decomposition and led to good yields of the desired products. Racemic 2-alkylmalonyl diastereomers were found to be separable by HPLC. The chiral stability of peptides containing optically active malonyl residues was investigated under simulated physiological conditions. Synthetic considerations for the incorporation of gem-diaminoalkyl and 2-alkylmalonyl residues into larger peptides to yield partially modified retro-inverso peptide analogs are presented.     

Synthesis of Met‐enkephalin by solution‐phase peptide synthesis methodology utilizing para‐toluene sulfonic acid as N‐terminal masking of l‐methionine amino acid

The Met‐enkephalin, Tyr‐Gly‐Gly‐Phe‐Met, was synthesized by the solution‐phase synthesis (SPS) methodology employing ‐OBzl group as carboxyls' protection, while the t‐Boc groups were employed for the N‐terminal α‐amines' protection for the majority of the amino acids of the pentapeptide sequence. The l ‐methionine (l ‐Met) amino acid was used as PTSA.Met‐OBzl obtained from the simultaneous protection of the α‐amino, and carboxyl group with para‐toluene sulfonic acid (PTSA) and as‐OBzl ester, respectively in a C‐terminal start of the 2 + 2 + 1 fragments condensation convergent synthetic approach. The protection strategy provided a short, single‐step, simultaneous, orthogonal, nearly quantitative, robust, and stable process to carry through the protected l ‐methionine and l ‐phenylalanine coupling without any structural deformities during coupling and workups. The structurally confirmed final pentapeptide product was feasibly obtained in good yields through the current approach.