Applications of BOP reagent in solid phase synthesis Advantages of BOP reagent for difficult couplings exemplified by a synthesis of [Ala 15]-GRF(1–29)-NH2

The BOP reagent [benzotriazol-l-yl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate] 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 (<10). The use of the BOP reagent with unprotected Tyr in solid phase peptide synthesis was also clearly established.     

Cyclization of peptides on a solid support. Application to cyclic analogs of Substance P

The general conditions for cyclization of peptides on polymer matrix by disulfide bridge formation are reported. This procedure is based on attack of 3-nitro-2-pyridinesulfenyl group (Npys) by a thiol function. It has been used for synthesis of five cyclic analogs of Substance P.     

Applications of BOP reagent in solid phase synthesis

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 N^α-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.     

Synthesis,biological activity and conformational analysis of cyclic GRF analogs

A novel cyclic GRF analog, cyclo(Asp⁸-Lys¹²)-[Asp⁸,Ala¹⁵]-GRF(1-29)-NH₂, i.e. cyclo^8.12[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 (～2h) using the BOP reagent. Substitution of Ala¹² with d -Ala² and/or NH₂-terminal replacement (desNH₂-Tyr¹ or N-MeTyr¹) in the cyclo^8.12[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 cyclo^8.12[Asp⁸,Ala¹⁵]-GRF(1-29)-NH₂ contains a long α-helical segment even in aqueous solution. A series of cyclo^8.12 stereoisomers containing d -Asp⁸ and/or d -Lys¹² were prepared and also found to be highly potent and to retain significant α-helical conformation. The high biological activity of cyclo^8.12[N-MeTyr¹,d -Ala²,Asp⁸,Ala¹⁵]-GRF(1-29)-NH₂ may be explained on the basis of retention of a preferred bioactive conformation.     

Synthesis of side-chain to side-chain cyclized peptide analogs on solid supports

Cyclic peptide structures of the type -Lys-R₁—R_n-Glu- can be synthesized on the Merrifield resin by assembling the peptide chain using Nα-Fmoc-amino acids and Boc and tert.-butyl protection for the side-chains of Lys and Glu, respectively. If residues R₁ to R_n contain side-chain functional groups, TFA-resistant protection is required. After TFA treatment cyclization on the resin can be performed with appropriate coupling reagents. The formation of such cyclic structures may be preceded or followed by peptide chain assembly using Nα-Boc-amino acids and the entire peptide chain containing the cyclic portion is finally cleaved by HF treatment. Using this principle we synthesized the following opioid peptide related cyclic analogs: H-Tyr-d -Lys-Gly-Phe-Glu-NH₂ (I), H-Tyr-Lys-Gly-Phe-Glu-NH₂ (II), H-Tyr-d -Lys-Phe-Glu-NH₂ (III), H-Tyr-d -Glu-Gly-Phe-Lys-NH₂ (IV), H-Tyr-d -Glu-Phe-Lys-NH₂ (V), H-Tyr-d -Orn-Gly-Glu-NH₂ (VI) and H-Tyr-d -Ala-Lys-Phe-Glu-NH₂ (VII). Cyclic monomers were obtained in all cases, as demonstrated by mass spectrometry. Analysis of side-products revealed a slow-down of the HF deprotection of O-benzylated tyrosine as a consequence of hydrophobic interactions as well as the formation of a side-chain-linked antiparallel cyclic dimer in the case of compound VI. In conclusion, the described method permits the convenient preparation of peptide analogs cyclized via amide bond formation between side-chain amino and carboxyl groups in reasonable yield.     

Applications of BOP reagent in solid phase peptide synthesis III. Solid phase peptide synthesis with unprotected aliphatic and aromatic hydroxyamino acids using BOP reagent

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.     

Synthesis of homologous peptides using fragment condensation: analogs of an HIV proteinase substrate

Two protected peptides Boc-Val-Ser(Bzl)-Gln-Asn-Tyr(BrZ)OH and Boc-Val-Ser(Bzl)-Gln-Asn-Tyr(BrZ)-ProOH were synthesized on a resin substituted by 9-(hydroxymethyl)-2-fluoreneacetic acid. After cleavage with piperidine/DMF, desalting, and activation, these peptides were used for the synthesis of 11 analogs of an HIV proteinase nonapeptide substrate Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-NH₂ using fragment condensation in solid phase. The fragment condensation was made in an ultrasonic bath. Using only 2 equivalents of the activated peptide in a DMF solution, this reaction was complete in 2h. All nonapeptides were assayed as substrates for HIV-1 and HIV-2 proteinases.     

Repetitive BOP coupling (REBOP) in solid phase peptide synthesis

The coupling reagent (benzotriazol-1-yloxy)tris-(dimethylamino)phosphonium (BOP) hexafluorophosphate was tested in the synthesis of luliberin (LH-RH) with inexpensive classically protected Boc-amino acids, in slight excess, and benzhydryl amino resin, without any other additive. The good solubility of this reagent and its by-products is of particular interest for automated peptide synthesis. [D-H-S¹]LH-RH was also synthesized and compared with LH-RH by proton nuclear magnetic resonance spectroscopy. As shown by the biological tests and the high performance liquid chromatography study, unprotected pyroClu and Boc-His can be used without any significant racemization but Boc-His(Boc) was found to be preferable since it gave no detectable racemization and no by-products. The difficult isolation of the minority D-derivative from the crude preparation of LH-RH was resolved by a recycling procedure in reversed phase HPLC.     

BOP reagent for the coupling of pGlu and Boc-His(Tos) in solid phase peptide synthesis

The model peptide TRH was successfully synthesized using benzotriazol-l-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.     

Synthesis and backbone conformations of cyclic hexapeptides cyclo-(Xxx-Pro-D-Gln)2

The solution syntheses of cyclo-(Xxx-Pro-D-Gln)₂, where Xxx=Gly, Ala, Leu, Phe and Val are described. Several routes were examined, the most successful involving the intermediate Z-Xxx-Pro-D-Gln-O-tBu and proceeding to cyclization of H-Xxx-Pro-D-Gln-Xxx-Pro-D-Gln-OH using diphenylphosphoryl azide. The N-H regions of the proton magnetic resonance spectra of aqueous solutions of these peptides were examined, and in the Xxx=Leu and Val peptides an unsymmetrical backbone, presumably with one cis Xxx-Pro peptide bond, was found to be important. Previous reports of cyclo-(Xxx-Pro-D-Yyy)₂ peptides have shown only C₂-symmetric forms.     

Asparagine coupling in Fmoc solid phase peptide synthesis

To investigate side reactions during the activation of side chain unprotected asparagine in Fmoc-solid phase peptide synthesis the peptide Met-Lys-Asn-Val-Pro-Glu-Pro-Ser was synthesized using different coupling conditions for introduction of the asparagine residue. Asparagine was activated by DCC/HOBt, BOP (Castro's reagent) or introduced as the pentafluorophenyl ester. The resulting peptide products were analyzed by HPLC, mass spectrometry and Edman degradation. In the crude products varying amounts of β-cyano alanine were found, which had been formed by dehydration of the side chain amide during carboxyl activation of Fmoc-asparagine. A homogeneous peptide was obtained by using either side chain protected asparagine derivatives with BOP mediated activation or by coupling of Fmoc-Asn-OPfp. Fmoc-Asn(Mbh)-OH and Fmoc-Asn(Tmob)-OH were coupled rapidly and without side reactions with BOP. For the side chain protected derivatives the coupling was as fast as that of other Fmoc-amino acid derivatives, whereas couplings of Fmoc-Asn-OH proceed more slowly. However, during acidolytic cleavage both protection groups, Mbh and Tmob, generate carbonium ions which readily alkylate tryptophan residues in a peptide. Tryptophan modification was examined using the model peptide Asn-Trp-Asn-Val-Pro-Glu-Pro-Ser. Alkylation could be reduced by addition of scavengers to the TFA during cleavage and side chain deprotection. A homogeneous peptide containing both, asparagine and tryptophan, was obtained only by coupling of Fmoc-Asn-OPfp.     

Synthesis of carbon‐14–labelled peptides

Carbon‐14 (¹⁴C)–labelled active pharmaceutical ingredients (APIs) and investigational medicinal products (IMPs) are required for phase 0/I to phase III mass balance and micro‐dosing clinical trials. In some cases, this may involve the synthesis of ¹⁴C‐labelled peptides, and the analysis can be performed by accelerated mass spectrometry (AMS). The ¹⁴C‐peptide is typically prepared by the solid‐phase peptide synthesis (SPPS) approach using custom‐made glassware for the key coupling steps. Further modification of the purified ¹⁴C‐peptide can then be performed.     

Incorporation of azaglutamine residues into peptides synthesised by the ultra-high load solid (gel)-phase technique

The solid (gel)-phase peptide synthesis of peptides, each containing an azaglutamine residue, has been examined. Procedures using various mono-, di- and tripeptide and carbazate fragments containing or relating to an azaglutamine (1) residue have been evaluated. N-Activation of the amino-terminus of a resin-bound peptide with bis-(2,4-dinitrophenyl)carbonate (2) yielded the terminal isocyanate species, which reacted with protected carbazates to give resin-bound protected peptides containing the aza-residue. By contrast, coupling of activated amino-acid derivates to the free amino-group of a resin-bound peptide with an aza-residue at the N-terminus was a slow and unsatisfactory process. It is concluded that the route yielding the best results involves the reaction of a protected amino-acyl carbazate to a resin-bound isocyanate-activated peptide.     

Acid-labile anchoring linkages for solid phase synthesis of C-terminal asparagine peptides using the Fmoc strategy

Two acid-labile substituted benzylamine type anchoring linkages, 4-benzoxy-2,6-dimethoxybenzylamine and 2-benzoxy-4,6-dimethoxybenzylamine, for solid phase synthesis of peptide amides were prepared. The N^a-9-fluorenylmethyloxycarbonyl (Fmoc) amino acids could be easily attached to the resins with DCC/HOBt (loading 0.5–0.6 mmol/g resin). After final removal of the N^a-protecting groups, treatment with TFA (50–95%) yielded amino acid and peptide amides in high purity. As we could show for the synthesis of thymulin (FTS, pGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn), these two resins with anchoring linkages are well suited for the synthesis of C-terminal Asn peptides using protected aspartic acid derivative as starting material.     

A comparative study of cyclization strategies applied to the synthesis of head-to-tail cyclic analogs of a viral epitope

A family of head-to-tail cyclic peptide models of the antigenic site A (G-H loop of viral protein 1) of foot-and-mouth disease virus has been designed on the basis of the three-dimensional structure adopted by the linear peptide YTASARGDLAHLTTT upon binding to neutralizing monoclonal antibodies. Three different methods of cyclization have been examined to access the peptides. Solution cyclization of a minimally protected linear precursor provided the expected products but required several purification steps that lowered the yields to 10%. The two other approaches relied on side-chain anchoring of the peptide through the Asp residue and cyclization on the solid phase. A synthetic scheme combining Fmoc, tBu and OAI protections was practicable but inefficient when scaled-up. The combination of Boc, Bzl and OFm protections was more promising, but suffered from high epimerization during the initial esterification of Boc-Asp-OFm to benzyl alcohol-type resins. This problem was solved by performing the esterification via the cesium salt of Boc-Asp-OFm. With this improvement, the Boc/Bzl/OFm has become the method of choice for the preparation of cyclic head-to-tail peptides in satisfactory yields and with minimal purification.     

Synthesis and properties of cyclic peptides containing the activation site of plasminogen

The activation of plasminogen results from proteolytic cleavage of the Arg₅₆₀-Val₅₆₁ bond by plasminogen activators (Sottrup-Jensen et al. PNAS (1975) 72, 2577). This region of the zymogen occurs in a small disulfide loop that must restrict the conformation around this bond. The nonapeptide sequence NH₂-Cys-Pro-Gly-Arg-Val-Val-Gly-Gly-Cys-NH₂ of plasminogen containing the activator sensitive arginyl valine bond was synthesized by carbodiimide coupling of Boc-Cys-Pro-Gly-OH(S-4-methylbenzyl) to NH₂-Arg(NO₂)-Val-Val-Gly-Gly-Cys-NH₂(S-4-methylbenz(S-4-methylbenzyl), followed by HF treatment and K₃Fe(CN)₆ oxidation to form a disulfide bond. Purified peptide was not a substrate for urokinase (UK) or plasminogen activator (PA) but possessed a slightly inhibitory activity towards PA. Addition of a lysine to the N-terminus of the nonapeptide yielded a decapeptide sequence of plasminogen that was a better substrate for UK but not for PA. The decapeptide inhibits PA slightly but not UK. These results suggest that active site geometry for PA must be more restrictive than that of UK and that other regions may be involved in the productive interactions with the activators inducing a better fit of the cyclic peptide loop.     

Synthesis of support-bound peptides carrying color labels

In order to check the possibility of labeling sub-libraries, we synthesized a colored support by acylation of aminomethyl resin with a special Boc-amino acid (a red azo dye). Syntheses of model peptides proved that the colored resin is suitable for solid phase peptide synthesis. Color labeling was also made by coupling resin-bound peptides at their N-termini with a blue dye. Colored resins as well as labeling at the N-terminus offer a possibility to simplify the preparation of support-bound peptide sub-library kits and their use in sequence determination of bioactive peptides. © 1994 Wiley-Less, Inc.     

Synthesis of O-phosphonotyrosyl peptides

A general method for the synthesis of O-phosphonotyrosyl peptides using solid phase methodology is described. Protected O-phosphonotyrosine derivatives with the general structure Boc-Tyr(R₂PO₃)-OH (R = methyl, ethyl or benzyl) were prepared as potential synthons for the introduction of O-phosphonotyrosine residues into peptide sequences. Using ³¹P n.m.r. spectroscopy, the alkyl phosphate protecting groups (R = methyl or ethyl) were shown to be stable to the coupling, deprotection and neutralization cycles of the Merrifield method of solid phase peptide synthesis. Facile removal of the methyl phosphate protecting groups from the O-phosphonotyrosyl peptide analogue Ac-Tyr(Me₂PO₃)-NHMe was demonstrated using 45% HBr/acetic acid. The O-phosphonotyrosyl heptapeptide H-Leu-Arg-Arg-Ala-PTyr-Leu-Gly-OH was subsequently prepared using solid phase methodology via incorporation of N²-tert-butyloxycarbonyl-O-dimethylphosphonotyrosine.     

A novel Fmoc-based anchorage for the synthesis of protected peptides on solid phase

A novel bifunctional compound, 9-(hydroxymethyl)-2-fluoreneacetic acid, was synthesized, coupled to benzhydrylamine-resin, and evaluated for its application to the solid phase synthesis of protected peptide fragments. Anchor-bond cleavage was achieved with 15% pipetidine/DMF. A protected heptapeptide, Boc-Val-Val-Ser(Bzl)-His(Tos)-Phe-Asn-Lys-(Z)-OH, corresponding to the sequence (1–7) of rat-transforming growth factor-α, was synthesized using this new support with an overall yield of 46%.     

The Chemical Shift Index method applied to resin‐bound peptides

Abstract: The Chemical Shift Index (CSI) method proposed by Wishart et al. [Biochemistry (1992) 31 , 1647–1651] to evaluate the secondary structure of peptides in aqueous solution uses as its reference the chemical shift values of each of the 20 natural amino acids (X) in a typical nonstructured sequence GGXAGG (17–20). In order to apply the CSI method to protected resin‐bound peptides, we established a new database of chemical shift values for the same GGXAGG sequences in their protected form and anchored to a polystyrene resin swollen in DMF‐d₇. The predictive value of this new reference set in the CSI protocol was tested on different resin‐bound peptides that were previously characterized by a full NOE analysis.