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An efficient, highly chemoselective PdCl2(PPh3)2 catalyzed, C–C bond formation of tautomerizable quinolinones with various boronic acids via C–OH bond activation using benzotriazol-1-yloxytris(dimethylamino) phosphonium hexafluorophosphate (BOP) reagent in the presence of K2CO3/1,4-dioxane system under aqueous condition, leads to the formation of functionalized quinolines in excellent yields, which offers great utility advantages in the synthesis of interesting compounds.

The model peptide TRH was successfully synthesized using benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent). The coupling reactions were carried out in N,N-dimethylformamide or N-methylpyrrolidone. These solvents allowed the incorporation of the N-terminal pyroglutamic acid residue into the peptide chain, without using the derivative bearing the N-benzyloxycarbonyl group, which acts as a solubility promoter. A comparative racemization study showed that Boc-His(Tos) can be coupled by means of BOP reagent with less racemization than with DCC when the amount of diisopropylethylamine (DIEA) is kept minimal (same ratio of equivalents as for Boc-His(Tos), i.e. 3 equiv.). However, with the use of a larger amount of DIEA in the coupling mixture (9 equiv.), approximately 3% of epimer was found in the crude product. Our study showed that even under low DIEA conditions, the rate of coupling of the residues with BOP remained comparable to that observed with DCC.

Using a variety of activating agents, a kinetic study was carried out to evaluate the rate of solid phase side-chain to side-chain cyclization of Asp3 to Lys12 in the model peptide-resin, [Ala15]-GRF(1–29)-BHA-resin. Asp3 and Lys12 were introduced in the peptide chain by using Nα-Boc amino acids in conjunction with the OFm/Fmoc side-chain protection scheme. The OFm and Fmoc side-chain protecting groups were shown to be stable to diisopropylethylamine and selectively deprotected on treatment with 20% piperidine in DMF. Solid phase side-chain to side-chain cyclization (lactamization) was shown to proceed to completion within 2 h using benzotriazol-1-yl-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate (BOP reagent) while the reaction was only 55% completed in 24h using DCC/HOBt. Solid phase side-chain to side-chain cyclization by the BOP procedure not only proceeded more rapidly but also gave a purer cyclic product.

A novel cyclic GRF analog, cyclo(Asp8-Lys12)-[Asp8,Ala15]-GRF(1-29)-NH2, i.e. cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2, was synthesized by the solid phase procedure and found to retain significant biological activity. Solid phase cyclization of Asp8 to Lys12 proceeded rapidly (approximately 2 h) using the BOP reagent. Substitution of Ala2 with D-Ala2 and/or NH2-terminal replacement (desNH2-Tyr1 or N-MeTyr1) in the cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2 system resulted in highly potent analogs that were also active in vivo. Conformational analysis (circular dichroism and molecular dynamics calculations based on NOE-derived distance constraints) demonstrated that cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2 contains a long alpha-helical segment even in aqueous solution. A series of cyclo8,12 stereoisomers containing D-Asp8 and/or D-Lys12 were prepared and also found to be highly potent and to retain significant alpha-helical conformation. The high biological activity of cyclo8,12[N-MeTyr1,D-Ala2,Asp8,Ala15]-GRF(1-29)- NH2 may be explained on the basis of retention of a preferred bioactive conformation.

A new improved synthesis of two immunostimulating peptides: FK-156 (D-lactyl-alanyl-gamma-D-glutamyl-(L)-meso-2,6-diaminopimelyl-(L)- glycine) and FK-565 (heptanoyl-gamma-D-glutamyl-(L)-meso-2,6-diaminopimelyl-(L)- D-alanine) is described. A proper differentiation between the two chiral amino acid moieties of diaminopimelic acid was accomplished by selective enzymatic hydrolysis of one methyl ester group of the L-centre of Z2-meso-A2pm(OMe)2 (2). Utilization of a commercially available protease and diester 2 as an enzyme substrate made possible the relatively simple synthesis of a key intermediate 4 and considerably simplified the final deprotection steps. Aminolysis of the N-carboxyanhydride (4) with D-AlaONBzl or GlyONBzl was chosen to obtain the appropriate dipeptides with one free amino group as convenient intermediates for further peptide synthesis. The BOP reagent, used for peptide bond formation, secured good yields and high chemical and chiral purity of the peptides. A modification of alanine deamination procedure leading to a significant increase of D-Lac(OAc) yield is presented.

In order to mimic a well-known loop structure (site A) of the hemagglutinin of influenza virus, a series of cyclic peptides derived from the region 139-147 were synthesized. The lactam analogs cyclised between the N-terminus Cys 139 and the beta-carboxyl of aspartic acid 148 (small loop) or the epsilon-NH2 of lysine 148 via succinimidyl linker (large loop) were synthesized by the solid phase method. Cyclisation was directly performed on the solid support prior to final cleavage of the peptide. We describe two protection schemes which allow us to obtain different loop sizes derived from the same sequence. Eight of the analogs contained relatively large ring structures (up to 38 membered). For protection of the side chain of aspartic acid in combination with N-alpha-Fmoc protection, the cyclohexyl ester was more satisfactory than the benzyl ester with respect to imide formation. When the rate of cyclodimerisation, as a function of resin substitution, was compared to the rate of cyclic monomer formation, it was found that dimerisation was proportional to the charge of the resin. Furthermore, a comparison of the recently reported BOP reagent over the classical DIPC/HOBt method for the cyclisation reaction shows that in our case the reaction proceeded more rapidly by the BOP procedure although it gave a less pure crude product.

The BOP reagent [benzotriazol-l-yl-oxy-tris-(dimethylamino)phosphonium hexa-fluorophosphate] introduced by Castro et al. [Tetrahedron Lett. (1975) 14, 1219-1222] is ideally suited for solid phase peptide synthesis. The rate of coupling using BOP compared favorably to DCC and other methods of activation including the symmetrical anhydride and DCC/HOBt procedures. BOP couplings using the solid phase procedure proceeded more rapidly and to a greater degree of completion for peptide bond formations that were previously determined to be very slow using the conventional DCC method. Stepwise solid phase peptide synthesis using BOP was successfully utilized for the preparation of the (22-29) and (13-29) fragments of [Ala15]-GRF(1-29)-NH2. Single couplings with 3 equiv. BOP and Boc-amino acids and 5.3 equiv. of diisopropylethylamine in DMF were used for each cycle. The yields of the fragments were superior and the purities comparable using the BOP procedure (single couplings) to those observed using multiple couplings via the DCC coupling method. A total synthesis of [Ala15]-GRF(1-29)-NH2 was also carried out using the BOP procedure (single couplings and 3 equiv. BOP and Boc-amino acids and 5.3 equiv. diisopropylethylamine in DMF for each cycle). Multiple couplings were only required for Boc-Asn-OH due to the proposed formation of Boc-aminosuccinimide during activation. The resultant GRF(1-29) analog was comparable to a control prepared with multiple DCC couplings under optimized conditions. In a parallel study, unprotected Boc-(hydroxy)-amino acids were successfully coupled with the BOP reagent. However, the number of coupling cycles after the introduction of unprotected hydroxy-amino acid must be minimal (less than 10). The use of the BOP reagent with unprotected Tyr in solid phase peptide synthesis was also clearly established.

The BOP reagent [benzotriazol-1-yl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate], which has been shown to be ideally suited for solid phase synthesis, has now been found to be useful for solid phase synthesis using a minimal side-chain protection scheme. This new application of the BOP reagent was exemplified by the successful synthesis of the CCK-7 analog, Ac-Tyr(SO3H)-Met-Gly-Trp-Met-Thr(SO3H)-Phe-NH2, using unprotected Boc(hydroxy)-amino acids [Boc-Thr-OH and Boc-Tyr-OH]. N-Terminal acetylation was achieved under mild conditions by using the BOP coupling reaction with acetic acid. This procedure provided the unprotected (Tyr27, Thr32)-peptide-resin which is ready for the required sulfation on the solid support without selective side-chain deprotection of Ty27 and Thr32. Solid phase sulfation was evaluated under a variety of conditions and it was determined that disulfation was optimal using pyridine acetyl sulfate (38 equiv.) in pyridine at 45° for 4 h. Shorter reaction times or milder conditions lead to the formation of the Thr32 monosulfated analog. Cleavage of the disulfated analog from the PAM-resin was achieved using liquid ammonia and the product was purified by preparative hplc and fully characterized. The advantages of the new procedure are compared with the reported synthesis of CCK-7.

Cyclic peptides cyclo(-Gly-Asp-Glu-Lys-), cyclo(-Gly-Gly-Asp-Glu-Lys-) and cyclo(-Gly-Gly-Gly-Asp-Glu-Lys-) were synthesized as models of theβ-turn of nerve growth factor loop 4. The corresponding protected linear precursors were obtained in 52–83% yields by the solid-phase method with the use of the Fmoc/But strategy and a chlorotrityl anchor group. The cyclization was carried out with benzotriazolyloxytris(dimethylamino)phosphonium (BOP) hexafluorophosphate, N-[(1H-benzotriazole-1-yl)-(dimethylamino)methylene]-N-methylmetanaminium-N-oxide (HBTU) hexafluorophosphate, and diphenylphosphorylazide (DPPA) at a dilution of 10−3 M. The distribution of reaction products was studied for each cyclopeptide in dependence on the type of the coupling agent. The use of DPPA was shown to completely inhibit the formation of cyclodimers in the synthesis of five-and six-membered cyclopeptides; however, in the case of a four-membered peptide, an additional tenfold dilution of the reaction mixture was necessary to achieve the effect. The identification of several byproducts during the synthesis showed that the elongation of the polypeptide chain using the BOP reagent can be complicated by substantial racemization, and the cleavage of the chlorotrityl anchor group by 0.5% TFA in dichloromethane proceeds with insufficient selectivity and is accompanied by the premature Boc deblocking of the lysine side function.

An efficient, highly chemoselective PdCl2(PPh3)2 catalyzed, C–C bond formation of tautomerizable quinolinones with various boronic acids via C–OH bond activation using benzotriazol-1-yloxytris(dimethylamino) phosphonium hexafluorophosphate (BOP) reagent in the presence of K2CO3/1,4-dioxane system under aqueous condition, leads to the formation of functionalized quinolines in excellent yields, which offers great utility advantages in the synthesis of interesting compounds.

The ready access to a new class of carbohydrate mimetics was demonstrated by the synthesis of tetrameric carbopeptoids, in which glycosidic bonds were replaced with amide linkages. We herein describe the detailed synthetis method of β(1→2)- and β(1→6)-linked carbopeptoids starting from each d -glucosamine and d -glucose derivative. The building blocks were polymerized using BOP reagent and DIEA to form a homooligomer. These produced carbopeptoids are resistant to glycosidases and have interesting biological activity. With conformational analysis by molecular modeling calculation, β(1→2)-linked decamer showed a typical 16-helix form as a mimic of β-peptide. Therefore, our polysaccharide analogues have potential as peptide foldamers.

Cyclic homooligomers of a thymidine-based nucleoside amino acid were synthesized from the linear dimer using BOP reagent in the presence of DIPEA under dilute conditions. Conformational analysis by NMR and constrained MD studies revealed that all the cyclic products had symmetrical structures. The NH and CO groups in these molecules point in opposite directions with near perpendicular orientation with respect to the plane of the macrocyclic ring having CO on the same side as the base.

4-Amino-3-(aminomethyl)benzoic acid (1) (AmAbz) is a novel unnatural amino acid with promise in applications as a building block for the synthesis of peptidomimetics and as a scaffold for combinatorial chemistry. It was efficiently synthesized in three steps (63% overall yield) from 4-aminobenzoic acid, by means of regioselective amidomethylation with hydroxymethylphthalimide. AmAbz (1) contains three distinct functionalities which could be discriminated from one another. Firstly, Boc2O or Fmoc−OSu reacted selectively with the benzylamino group to give the monoprotected derivatives AmAbz(Boc) (8a) or AmAbz(Fmoc) (8b), respectively. The absence of acylation at the arylamino group was also noticed in coupling experiments using the BOP reagent and building block 8b. This made protection of the arylamino group unnecessary either for peptide bond formation at the carboxyl group, or for subsequent elongation of a peptide chain at the benzylamino group. Finally, the arylamino group could be acylated under base-free, carbodiimide-mediated coupling conditions. These properties are illustrated by the solid-phase synthesis of the AmAbz-containing branched pseudopeptide Fmoc−Ala−Phe−AmAbz(H−Lys−Leu)−Val−Gly−NH2 (15). The synthesis of Fmoc−AmAbz(Boc) (10) is also described.

A series of poly(ethylene glycol)−poly(l-lysine) block copolymers partially substituted in varying degrees with a hydrocinnamoyl group at the Ne-position was prepared by a coupling reaction of hydrocinnamic acid to the e-amino group of the poly(l-lysine) segment of the block copolymer using (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP Reagent) as a condensation reagent. The Ne-substituted block copolymers with less than 90% substitution formed clear solutions in 0.1 M phosphate buffer (pH 7.4) by dialyzing from DMSO. Light scattering measurements revealed multimolecular micelle formation for the block copolymers with 65−70% of the substitution degree. The cumulant diameter of the micelles was approximately 40 nm with a moderate polydispersity (μ2/Γ2 ∼ 0.15). A fairly low critical association concentration (∼40 mg/L) was estimated using pyrene as a fluorescence probe molecule. At a pH as high as 11, the block copolymer with a substitution degree even as low as 50%, which ga...

Hydroxybenzotriazolyl esters, formed in situ from carboxylic acids and BOP reagent, react with sodium borohydride in THF to give alcohols in high yields. This method is convenient, rapid and chemoselective, with such functional groups as nitro, halide, nitrile, azido and ester being unaffected.

The experimental antitumor drug Edatrexate, labeled with 99% 13C at the 2-position of the glutamate acid group was required for 13C-magnetic resonance spectroscopy studies in biological media. Coupling of 2,4-diamino-4-deoxy-10-ethyl-10-deazapteroic acid with diethyl L-2-13C-glutamate as promoted by BOP reagent afforded Edatrexate (2-13C-glu) diethyl ester in 60% yield following purification by column chromatography. Saponification by aqueous NaOH in 2-methoxyethanol gave the target molecule in 44% yield or 26% overall. © 1997 John Wiley & Sons, Ltd.

The syntheses of the Fmoc-protected Peptide Nucleic Acid (PNA) monomer pentafluorophenyl esters of adenine (26), cytosine (23), guanine (29) and thymine (20), and their oligomerization are described. The Fmoc PNA backbone 1 is prepared as a stable hydrochloride salt. The base acetic acids of adenine (4) and cytosine (3) were prepared by Cbz protection of the exocyclic amino groups followed by alkylation with t-butylbromoacetate and subsequent acid hydrolysis of the t-butyl ester. Allylation of 6-chloro-2-aminopurine followed by acid hydrolysis, Cbz protection with N-(benzyloxycarbonyl)imidazole, ozonolytic cleavage, and oxidation afforded the Cbz-protected guanine acetic acid (5). The base acetic acids (2, 3, 4 and 5) were coupled to the backbone (1) with either EDC (2 and 3) or BOP reagent (4 and 5). Acid hydrolysis of the resulting t-butyl esters and transesterification afforded the corresponding pentafluorophenyl esters (20, 23, 26 and 29). Oligomerization is conducted on a 0.05 mmol scale with a mere 2 fold excess of monomer in each coupling cycle. The N-terminal Fmoc group is retained on the final oligomer, following HF cleavage and deprotection, providing a convenient lipophilic handle for HPLC purification.

Eight cyclic analogs and corresponding linear homologs of the alpha-factor mating pheromone (WHWLQLKPGQPMY) of Saccharomyces cerevisiae were synthesized using solid-phase procedures on a phenylacetamidomethyl support. On-resin lactamization of the side chains of residues 7 and 10 to form rings containing from 14 to 18 atoms was effected by the BOP reagent. All peptides were highly homogeneous and gave expected molecular ions by FAB mass spectrometry. The constrained analogs had biological activities varying from 10% to less than 0.1% of that of [Nle12]-alpha-factor. In all cases, cyclic analogs with Glu in position 10 were more active than the homolog with Asp at this position. This trend was also found with the corresponding linear pheromones, suggesting that a gamma-carbonyl in position 10 is an important determinant of pheromone potency. The cyclic peptides had from 50- to 20000-fold lower affinities for the alpha-factor receptor than for [Nle12]-alpha-factor, as judged using a competition binding assay. Circular dichroism studies indicate that the cyclic lactam-containing region of cyclo7.10[Orn7, Glu10,Nle12]-alpha-factor retains a beta-turn-like structure similar to that found in the corresponding model tetrapeptide. The results show that covalently constrained analogs of the linear pheromone can maintain biological activity, despite binding poorly to the receptor, and indicate that a beta-turn-like structure in the center of the pheromone allows signal transduction.

A simple procedure for preparation of esters under mild conditions employing the BOP reagent is reported. Acid and base labile protecting groups commonly used with amino acids e.g. t-butyl, Fmoc etc., are well tolerated under these conditions. The mechanism and scope of this reaction are briefly discussed.

A convenient synthetic method is described for the preparation of peptide-methotrexate (MTX) conjugates in which MTX is coupled selectively through the gamma-carboxyl group of its glutamic acid moiety to a free amino group in peptide analogs. The syntheses of a somatostatin analog-MTX conjugate (MTX-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2) (AN-51) and two conjugates of analogs of luteinizing hormone-releasing hormone (LH-RH) with MTX [Glp-His-Trp-Ser-Tyr-D-Lys(MTX)-Leu-Arg-Pro-Gly-NH2] (AJ-04) and [Ac-Ser-Tyr-D-Lys(MTX)-Leu-Arg-Pro-NH-Et] AJ-51 are presented as examples. Benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent) was used in the synthesis for activation of 4-amino-4-deoxy-N10-methylpteroic acid, which reacted with the potassium salt of glutamic acid alpha-tert-butyl ester in dimethyl sulfoxide to form the suitably protected MTX derivative. This synthesis provides an example of the high suitability of BOP reagent for the salt-coupling method. The selectively protected MTX derivative was then coupled to the different peptide carriers and deprotected under relatively mild conditions by trifluoroacetic acid. The conjugates of MTX with hormonal analogs are suitable for targeting to various tumors that possess receptors for the peptide moieties.

Vasoactive intestinal peptide (VIP) was assembled on a polyacrylamide gel using a combination of the Boc and Fmoc peptide synthesis strategies. Before the synthesis, the polymeric matrix functionalized with sarcosine methylester was treated with ethylenediamine in order to form primary amine reaction sites (0.3 mmol/g). Then a six-carbon spacer arm, Boc-aminocaproic acid, was coupled to the gel after activation with benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) reagent. After acidolysis of the Boc protecting group, the derivative Boc-asparaginyl (xanthenyl)-4-(oxymethyl)phenylacetic acid was introduced into the polyacrylamide resin. Leucine-27 and isoleucine-26 were incorporated into the peptide chain as Boc-protected derivatives while the subsequent amino acids were all introduced as Fmoc residues. All couplings were achieved with BOP reagent in presence of diisopropylethylamine. The synthesis proceeded easily and only asparagine-9 required a double coupling step. After completion of the VIP assemblage, the side-chain protecting groups were removed by reaction with trifluoroacetic acid containing appropriate scavengers. A sample of peptide-resin was treated with hydrofluoric acid and the quality of the synthetic VIP-COOH material, obtained after cleavage, was assessed by reverse-phase HPLC and fast atom bombardment mass spectrometry. The compatibility with aqueous solutions of the polyacrylamide resin loaded with VIP (0.15 mmol/g), as well as its ability to be utilized as an affinity matrix for VIP receptors, was demonstrated using solubilized receptor preparation made from porcine liver membrane. After a single-step affinity chromatography, no binding activity was anymore detectable in the pass-through fraction. Moreover, after elution of the bound material, a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed a single band with an M r of 53 kDa after either silver staining or radioiodination. The specificity of the affinity matrix was demonstrated by adding free VIP to the detergent extract prior to the loading onto the column. Under these conditions, no protein hand was detected after SDS-PAGE analysis.

A simple procedure for preparation of esters under mild conditions employing the BOP reagent is reported. Acid and base labile protecting groups commonly used with amino acids e.g. t-butyl, Fmoc etc., are well tolerated under these conditions. The mechanism and scope of this reaction are briefly discussed.

Hydroxybenzotriazolyl esters, formed in situ from carboxylic acids and BOP reagent, react with sodium borohydride in THF to give alcohols in high yields. This method is convenient, rapid and chemoselective, with such functional groups as nitro, halide, nitrile, azido and ester being unaffected.

International audience; Two cyclic peptides have been prepared and characterized by MS and NMR. The linear precursors have been obtained by solid phase synthesis and the cycliation step was achieved using the BOP reagent. They increase markedly the rate of hydrolysis ofSynthesis and characterization of two cyclic peptides are described. They increase markedly the rate of oligoribonucleotide hydrolysis. oligoribonucleotides.

BOP coupling reagent suppresses the formation of diketopiperazines in the solid-phase coupling of the third amino acid to dipeptides when a nitro-benzylic “handle” is used to link the peptide to the resin. The amino function of the second amino acid may be deprotected with TFA and the coupling carried out without a prior neutralisation step.

Until recently, large compound libraries produced by solid-phase synthesis have been restricted to those of linear peptides and oligonucleotides, but the need for development and synthesis of libraries of small organic molecules using this method is growing rapidly.1 We are interested in developing strategies and chemistries that would allow entry to combinatorial libraries of peptidomimetic structures on solid support. Our approach to the synthesis of peptide mimetics involves attachment of an essential amino acid residue via its side-chain functionality to the solid support. A similar strategy has been employed by Ellman in creating a peptidomimetic library using the hydroxyethylamino isostere of phenylalanine found in most HIV protease inhibitors anchored via the secondary alcohol.2 Anchoring the amino acid side chain to polymeric supports is now widely used to prepare head-to-tail cyclic peptides via solid-phase-mediated cyclization.3 Typically, Asp or Glu are attached via their ω-carboxyls to hydroxymethyl or aminomethyl resins, with the expectation that the corresponding peptides containing Asp/Glu or Asn/Gln, respectively, will be obtained after final cleavage from the resin. The most widely practiced strategy to obtain head-to-tail cyclic peptides uses the three-dimensional Fmoc/t-Bu/allyl orthogonal protection scheme. Kates et al. developed the use of R-allyl-protected aspartic acid for automated continuous flow synthesis of cyclic peptides containing Asp or Asn residues via side-chain anchoring to the resin followed by on-resin cyclization,4 which is applicable to Gluand Gln-containing sequences. Also, the Fmoc/allyl ester protection scheme was applied to anchor lysine via its -amino group.3e Additionally, side-chain attachment to Merrifield (chloromethylated polystyrene) resin has been applied to cysteine in its unprotected form; this has allowed both Nand C-terminal derivatization.5 In the latter case, C-terminal derivatization to amides and esters required carboxylic acid activation with BOP reagent.

Relatively few cyclic peptides have reached the pharmaceutical marketplace during the past decade, most produced through fermentation rather than made synthetically. Generally, this class of compounds is synthesized for research purposes on milligram scales by solid-phase methods, but if the potential of macrocyclic peptidomimetics is to be realized, low-cost larger scale solution-phase syntheses need to be devised and optimized to provide sufficient quantities for preclinical, clinical, and commercial uses. Here, we describe a cheap, medium-scale, solution-phase synthesis of the first reported highly potent, selective, and orally active antagonist of the human C5a receptor. This compound, Ac-Phe[Orn-Pro-d-Cha-Trp-Arg], known as 3D53, is a macrocyclic peptidomimetic of the human plasma protein C5a and displays excellent antiinflammatory activity in numerous animal models of human disease. In a convergent approach, two tripeptide fragments Ac-Phe-Orn(Boc)-Pro-OH and H-d-Cha-Trp(For)-Arg-OEt were first prepared by high-yielding solution-phase couplings using a mixed anhydride method before coupling them to give a linear hexapeptide which, after deprotection, was obtained in 38% overall yield from the commercially available amino acids. Cyclization in solution using BOP reagent gave the antagonist in 33% yield (13% overall) after HPLC purification. Significant features of the synthesis were that the Arg side chain was left unprotected throughout, the component Boc-d-Cha-OH was obtained very efficiently via hydrogenation of d-Phe with PtO(2) in TFA/water, the tripeptides were coupled at the Pro-Cha junction to minimize racemization via the oxazolone pathway, and the entire synthesis was carried out without purification of any intermediates. The target cyclic product was purified (97%) by reversed-phase HPLC. This convergent synthesis with minimal use of protecting groups allowed batches of 50-100 g to be prepared efficiently in high yield using standard laboratory equipment. This type of procedure should be useful for making even larger quantities of this and other macrocyclic peptidomimetic drugs.

Azomycin glucuronate was coupled to a PnAO ligand to create azomycin-based ligands that would form water-soluble 99mTc-azomycin complexes for imaging hypoxic tissue. 1-beta-D-(2-Nitroimidazolyl)glucuronic acid, (see structure in text), was synthesized by coupling 2-nitroimidazole with 1-alpha-bromo-2,3,4-tri-O-acetyl-6-methyl glucuronate, followed by deprotection. Reaction of (see structure in text) with 6-methyl-6-methylamino-HMPnAO (Pn-44) in the presence of BOP reagent in anhydrous dimethyl sulfoxide afforded the PnAO-glucuronides (see structure in text) and (see structure in text). Compound (see structure in text) was isolated in three rotomeric forms. Biological evaluation of (see structure in text) (99mTc-5) indicated selective binding to hypoxic EMT-6 cells, and cytotoxicity to fibroblasts and HeLa, sk24, sk23, and g361 cancer cell lines, at an IC20

The solid phase synthesis of a series of piperazinone-derived Leu-enkephalin analogues is presented. The initial step in the synthesis involved the N-alkylation of Wang resin bound N-(4-tert-butyloxy-phenethyl)-glycine with D or L Boc-serine-beta-lactone (the Vederas lactone). The resulting carboxylic acid was then coupled to a variety of monosubstituted benzylamine derivatives using benzotriazol-1-yloxy-tris(dimethyl-amino)phosphonium hexafluorophosphate (the BOP reagent) to yield a series of resin bound tertiary amides. Treatment with 5% H2O in TFA resulted in the facile cleavage, deprotection, and cyclization of this linear precursor to yield a series of piperazinones (compounds 1-8).

A new fluorigenic substrate for the determination of biotinidase, N-D-biotinyl-7-amino-4-methylcoumarin has been synthesized by using BOP reagent. This substrate was used for measuring the enzyme activity in plasma of normal and biotinidase deficient patients. The methodology used here should be applicable to other biological fluids and homogenates to detect biotinidase deficiency.

In an effort to explore the effect of ring size on the biologically active conformation of cyclic analogs of the mating pheromone α-factor (WHWLQLKPGQPMY) from Saccharomyces cerevisiae, eight cyclic tetrapeptides corresponding to the KPGQ portion of α-factor were synthesized. These N-α-acetyl/carboxyl amide terminal cyclic tetrapeptides were prepared on a 4-methylbenzhydrylamine resin using orthogonal Boc, Frnoc, OFm and OtBut protecting groups and HOBt-DIPC accelerated active esters or urethane-protected N-carboxyanhydrides. On-resin cyclization of the side-chain amino and carboxyl groups of the first and fourth residues, respectively, was performed with the BOP reagent to generate lactams containing 14–18 atoms. HF cleavage resulted in two products, the desired cyclic tetrapeptide and a major side product. All peptides were purified to near homogeneity (>99%) by using reversed-phase HPLC and were characterized by FABMS and 1H NMR. Certain constrained cyclic tetrapeptides appear to be a mixture of isomers at room temperature as evidenced by HPLC and NMR. The major side product has been identified as a cyclo dimer, obtained as a consequence of interchain cyclization on the resin. CD analysis in several solvents gives evidence that some of the cyclic tetrapeptides exist in β-turn conformations. © Munksgaard 1995.

The model peptide TRH was successfully synthesized using benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent). The coupling reactions were carried out in N,N-dimethylformamide or N-methylpyrrolidone. These solvents allowed the incorporation of the N-terminal pyroglutamic acid residue into the peptide chain, without using the derivative bearing the N-benzyloxycarbonyl group, which acts as a solubility promoter. A comparative racemization study showed that Boc-His(Tos) can be coupled by means of BOP reagent with less racemization than with DCC when the amount of diisopropylethylamine (DIEA) is kept minimal (same ratio of equivalents as for Boc-His(Tos), i.e. 3 equiv.). However, with the use of a larger amount of DIEA in the coupling mixture (9 equiv.), approximately 3% of epimer was found in the crude product. Our study showed that even under low DIEA conditions, the rate of coupling of the residues with BOP remained comparable to that observed with DCC.

An approach to the solid-phase segment condensation synthesis of the 17-peptide amide human gastrin-I has been developed. N alpha-amino and side-chain protection were provided by 9-fluorenylmethyloxycarbonyl (Fmoc) and tert.-butyl groups, and a series of anchors cleavable under mild conditions were used. The N-terminal pentapeptide pGlu-Gly-Pro-Trp-Leu-OH was prepared using a p-alkoxybenzyl ester linkage made by a preformed handle strategy. Cleavage, in 65% yield, was with the new Reagent M: CF3COOH-CH2Cl2-beta-mercaptoethanol--anisole (70:30:2:1), which was optimized to preserve the labile tryptophan residue. A new preformed handle procedure expedited solid-phase synthesis of the protected "middle" hexapeptide, Fmoc-(Glu(OtBu]5-Ala-OH, anchored as an o-nitrobenzyl ester. Chains were not lost during this assembly, and final photolytic cleavage (350 nm) in toluene--CF3CH2OH (4:1) occurred in 59% yield. Both protected intermediates were purified by simple gel filtration, whereupon they were shown to be pure by analytical HPLC, and gave satisfactory NMR and FABMS spectra. Last, the C-terminal hexapeptide, Tyr(tBu)-Gly-Trp-Met-Asp(OtBu)-Phe, was assembled on a tris(alkoxy)benzylamide "PAL" support. For the polymer-supported segment condensation, the middle and N-terminal pieces were added respectively in greater than 98% and 89% yields (judged by amino acid analysis and solid-phase sequencing), by overnight couplings in N,N-dimethylformamide (DMF) mediated by benzotriazolyl N-oxytrisdimethylaminophosphonium hexafluorophosphate (BOP) in the presence of 1-hydroxybenzotriazole (HOBt) and N-methylmorpholine (NMM). Racemization was 4% and 11% respectively at Ala and Leu. Cleavage with Reagent M followed by reversed-phase chromatography gave pure gastrin-I in an overall 30% isolated yield. These results compare favorably with those from a stepwise assembly.

p-Hydroxyphenyl propionic resin (Table I, compound 4) was used to prepare the 1–6 protected fragment of LH-RH which was then condensed with “BOP” (benzotriazolyl N-oxytrisdimethylaminophosphonium hexafluorophosphate) as coupling reagent to the 7–10 residue synthesized on the same resin. Peptidylresin was divided into two aliquots in order to obtain LH-RH after aminolysis and treatment with liquid hydrogen fluoride, LH-RH-COOH after saponification followed by hydrogenation, or treatment with liquid hydrogen fluoride. The resulting hormones were rapidly purified by the sole means of two gel filtrations.

Esterification of N-protected α-amino acids was achieved via isopropenyl chlorocarbonate (IPCC) activation. In situ alcoholysis of the unstable mixed anhydride intermediate was catalyzed by 4-(dimethylamino)pyridine (DMAP). Competing isopropenyl ester formation was negligible when using methylene chloride as the solvent. A variety of esters from primary and secondary alcohols were obtained with good yields (60 to 96 %), and even the more hindered tertiobutyl alcohol gave acceptable yields under more drastic conditions. The improvement in depsipeptide synthetic methodology is illustrated by preparation of the antibiotic valinomycin, using IPCC for ester bond formation, and BOP reagent for peptidic coupling and the last-step cyclization.

Benzotriazolyl-N-oxytris(dimethylamino)phosphonium hexafluorophosphate (‘Le BOP’ reagent) in combination with imidazole provides a very mild system for selective esterification of polyhydroxy-compounds. An example is given showing an improved selectivity towards position 2 of trehalose, leading to new derivatives of ‘Cord-Factor.’

A New Synthesis of Thymosin α1 Thymosin α1, an N-acetylated octacosa thymus peptide, isolated first from thymosin fraction 5, is synthesized by the solid phase method using the p-benzyloxybenzyl alcohol/polystyrene/divinylbenzene resin, Nα -Fmoc-amino acids and those with tert-butyl or Boc side chain protection. All couplings are performed with the Bop reagent. The peptide is purified by a combination of gel chromatography and preparative HPLC, its purity checked by amino acid analysis and analytical HPLC, and the biological activity tested by the E-rosette assay.

A successful synthesis of Asp1-Arg2-Val3-Tyr4-IIe5-His6-Pro7-Phe6-His9-Leu10-Leu11-Tyr12-Tyr13-Ser14 has been achieved by the classical stepwise solid-phase method using 1% crosslinked Merrifield resin and benzotriazolyloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent) respectively as solid support and coupling reagent. The coupling protocol included in situ neutralization of the amino partners after Boc-deprotection and the coupling times seldom exceeded 30 min. For His6 and His9, Boc-His(Boc). DCHA was neutralized in situ and allowed to couple. The peptide, purified by reparative h.p.l.c., was obtained in 29% overall yield based on the first residue attached to the solid support.

Linear and cyclic analogues of cyclolinopeptide A (CLA) in which one or both phenylalanine residues in fragment Pro6-Pro7-Phe8-Phe9 were substituted by their sulfonated derivatives have been synthesized by SPPS method and cyclization with the BOP reagent. The peptides were examined for their immunosuppressive activity in the humoral and cellular immune response by PFC and DTH tests. All of the analogues retain some immunosuppressive activity of native CLA. Their CD spectra confirm that the optical activity of aromatic residues in CLA depends on their position in the peptide chain. Only the residue in position 8 seems to be optically active. CD spectrum of the cyclic analogue modified in position 9 is very similar to that of native CLA which correlates with its high biological activity. The chiroptical properties of the p-sulfonated Phe-residue are established.

A successful synthesis of Asp1-Arg2-Val3-Tyr4-IIe5-His6-Pro7-Phe6-His9-Leu10-Leu11-Tyr12-Tyr13-Ser14 has been achieved by the classical stepwise solid-phase method using 1% crosslinked Merrifield resin and benzotriazolyloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent) respectively as solid support and coupling reagent. The coupling protocol included in situ neutralization of the amino partners after Boc-deprotection and the coupling times seldom exceeded 30 min. For His6 and His9, Boc-His(Boc). DCHA was neutralized in situ and allowed to couple. The peptide, purified by reparative h.p.l.c., was obtained in 29% overall yield based on the first residue attached to the solid support.

The tetradecapeptide Ac-Asp1-Arg2-Val3-Tyr4-lle5-His6-Pro7-Phe8-His9-Leu10-Leu11-Val12-Tyr13-Ser14-OH has been synthesized by a three-segment coupling strategy. All the coupling steps were carried out with benzotriazolyloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent). Histidine was incorporated in good yield by using Boc(Nim-Boc)histidine (DCHA).