Heterotrimeric collagen peptides containing functional epitopes. Synthesis of single‐stranded collagen type I peptides related to the collagenase cleavage site

Synthetic collagen peptides containing larger numbers of Gly‐Pro‐Hyp repeats are difficult to purify by standard chromatographic procedures. Therefore, efficient strategies are required for the synthesis of higher molecular weight collagen‐type peptides. Applying the Fmoc/tBu chemistry, a comparative analysis of the standard stepwise chain elongation procedure on solid support with the procedure based on the use of the synthons Fmoc‐Gly‐Pro‐Hyp(tBu)‐OH and Fmoc‐Pro‐Hyp‐Gly‐OH was performed. The crude products resulting from the stepwise elongation procedure and from the use of Fmoc‐Gly‐Pro‐Hyp(tBu)‐OH clearly revealed large amounts of microheterogeneities that result from incomplete imino acid acylation as well as from diketopiperazine formation with cleavage of Gly‐Pro units from the growing peptide chain. Conversely, by the use of the Fmoc‐Pro‐Hyp‐Gly‐OH synthon, the quality of the crude products was significantly improved; moreover, protection of the Hyp side chain hydroxyl function is not required using the Fmoc/tBu strategy. With this optimized synthetic procedure, relatively large collagen‐type peptides were obtained in satisfactory yields as highly homogeneous compounds. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Racemisation of N‐Fmoc phenylglycine under mild microwave‐SPPS and conventional stepwise SPPS conditions: attempts to develop strategies for overcoming this

We have been engaged in the microwave‐solid phase peptide synthesis (SPPS) synthesis of the phenylglycine (Phg)‐containing pentapeptide H‐Ala‐Val‐Pro‐Phg‐Tyr‐NH₂ (1) previously demonstrated to bind to the so‐called BIR3 domain of the anti‐apoptotic protein XIAP. Analysis of the target peptide by a combination of RP‐HPLC, ESI‐MS, and NMR revealed the presence of two diastereoisomers arising out of the racemisation of the Phg residue, with the percentage of the LLLDL component assessed as 49%. We performed the synthesis of peptide (1) using different microwave and conventional stepwise SPPS conditions in attempts to reduce the level of racemisation of the Phg residue and to determine at which part of the synthetic cycle the epimerization had occurred. We determined that racemisation occurred mainly during the Fmoc‐group removal and, to a much lesser extent, during activation/coupling of the Fmoc‐Phg‐OH residue. We were able to obtain the desired peptide with a 71% diastereomeric purity (29% LLLDL as impurity) by utilizing microwave‐assisted SPPS at 50 °C and power 22 Watts, when the triazine‐derived coupling reagent DMTMM‐BF₄ was used, together with NMM as an activator base, for the incorporation of this residue and 20% piperidine as an Fmoc‐deprotection base. In contrast, the phenylalanine analogue of the above peptide, H‐Ala‐Val‐Pro‐Phe‐Tyr‐NH₂ (2), was always obtained as a single diastereoisomer by using a range of standard coupling and deprotection conditions. Our findings suggest that the racemisation of Fmoc‐Phg‐OH, under both microwave‐SPPS and stepwise conventional SPPS syntheses conditions, is very facile but can be limited through the use of the above stated conditions. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of cyclic peptides and peptide libraries on a new disulfide linker.

A new cysteine-based disulfide linker for Fmoc solid phase peptide synthesis was developed (Fmoc-Cys(3-mercapto-3-methylbutanoic acid)OPp) that allows the on-resin assembly and side chain deprotection of cyclic peptides. Model peptides and a cyclic peptide library of the structure [a-a-x-x-a-a-c] composed of D-amino acids were assembled and the synthesis and cleavage conditions studied. The best cyclization results were obtained with PyBOP/HOAt/diisopropylethyl amine. Racemization rates of the cysteine in the analyzed model sequences were between 5.2 and 12.3%. Cleavage of the disulfide bond was best carried out with DTT in 50% 2-propanol/100 mM ammonium bicarbonate. The cleaved peptides can be used directly in biological assays.     

Side Reaction with N-Carboxymethyl Amino Acids in the Synthesis of Backbone Cyclized Peptides

The use of N-carboxymethyl amino acids in the assembly of peptides with backbone cyclization can lead to diketopiperazine formation by intramolecular aminolysis which occurs despite the tert-butyl protection of the carboxy group. This undesired side reaction can be prevented by a very short deprotection time for the Fmoc group, by elongation of the N-carboxyalkyl chain or by forming the backbone (lactam) bridge before Fmoc removal, but not by the use of DBU or additives.     

Solid-phase synthesis of a range of O-phosphorylated peptides by post-assembly phosphitylation and oxidation.

A completely general method for the O-phosphorylation of peptides of any given composition using solid-phase methodology is described. Peptides were assembled using Fmoc amino acid active esters, with base used for Fmoc deprotection. Unprotected amino acid side chain hydroxyl groups were phosphitylated and oxidised at the end of the assembly using bis(benzyloxy)(diisopropylamino)phosphine and tert.-butylhydroperoxide respectively. TFA was used for final deprotection of the amino acid side chains and for simultaneous cleavage from the resin. The synthesis of O-phosphopeptides of up to 15 residues in length is described.     

Side Reaction with N-Carboxymethyl Amino Acids in the Synthesis of Backbone Cyclized Peptides

Summary The use ofN-carboxymethyl amino acids in the assembly of peptides with backbone cyclization can lead to diketopiperazine formation by intramolecular aminolysis which occurs despite thetert-butyl protection of the carboxy group. This undesired side reaction can be prevented by a very short deprotection time for the Fmoc group, by elongation of theN-carboxyalkyl chain or by forming the backbone (lactam) bridge before Fmoc removal, but not by the use of DBU or additives.     

The importance of the N-terminal beta-turn in bradykinin antagonists

Three peptides, B-10148 (Lys-1-Lys0-Arg1-Pro2-Hyp3-Gly4-Igl5-Ser6- DF5F7-Oic8; where Hyp is trans-4-hydroxyproline, Igl is alpha-(2-indanyl)glycine, F5F is 2,3,4,5,6-pentafluorophenylalanine and Oic is (3aS,7aS)-octahydroindole-2-carboxylic acid), B-10206 (DArg0-Arg1-Pro2-Hyp3-Gly4-Igl5-Ser6-DF 5F7-Nc7G8-Arg9; where Nc7G is N-cycloheptylglycine) and B- 10284 (Arg1-Pro2-Pro3-Gly4-Phe5-Thr6-DTic7-Oic8- NH2; where Tic is 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), were studied in detail by NMR spectroscopy in 60% CD3OH /40% H2O and modeled by a simulated annealing protocol to determine their solution structure. B-10148, an extremely potent BK B1 receptor antagonist with very high BK B2 receptor antagonist activity, despite lacking a C-terminal Arg, displayed an ideal type II beta-turn from Pro2 to Igl5, as well as a salt bridge between the guanidino group of Arg1 and the carboXylate group of Oic8. B-10206, the most potent B2 antagonist, also displayed an ideal type II beta-turn from Pro2 to Igl5 but secondary structure was not observed at the C-terminal end. The third peptide, B-10284, a des-Arg9 analog with a C-terminal amide and a very potent B2 antagonist, had no definite solution structure. The high activity of these peptides emphasizes the importance of the N-terminal beta-turn and the hydrophobic character at the C-terminus in determining the activity of bradykinin antagonists.     

Enzymatic inactivation of bradykinin by rat brain neuronal perikarya

1. Bradykinin (Bk; Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg8) inactivation by bulk isolated neurons from rat brain is described. 2. Bk is rapidly inactivated by neuronal perikarya (4.2 +/- 0.6 fmol/min/cell body). 3. Sites of inactivating cleavages, determined by a kininase bioassay combined with a time-course Bk-product analysis, were the Phe5-Ser6, Pro7-Phe8, Gly4-Phe5, and Pro3-Gly4 peptide bonds. The cleavage of the Phe5-Ser6 bond inactivated Bk at least five fold faster than the other observed cleavages. 4. Inactivating peptidases were identified by the effect of inhibitors on Bk-product formation. The Phe5-Ser6 bond cleavage is attributed mainly to a calcium-activated thiol-endopeptidase, a predominantly soluble enzyme which did not behave as a metalloenzyme upon dialysis and was strongly inhibited by N-[1(R,S)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate and endo-oligopeptidase A antiserum. Thus, neuronal perikarya thiol-endopeptidase seems to differ from endo-oligopeptidase A and endopeptidase 24.15. 5. Endopeptidase 24.11 cleaves Bk at the Gly4-Phe5 and, to a larger extent, at the Pro7-Phe8 bond. The latter bond is also cleaved by angiotensin-converting enzyme (ACE) and prolyl endopeptidase (PE). PE also hydrolyzes Bk at the Pro3-Gly4 bond. 6. Secondary processing of Bk inactivation products occurs by (1) a rapid cleavage of Ser6-Pro7-Phe8-Arg8 at the Pro7-Phe8 bond by endopeptidase 24.11, 3820ACE, and PE; (2) a bestatin-sensitive breakdown of Phe8-Arg9; and (3) conversion of Arg1-Pro7 to Arg1-Phe5, of Gly4-Arg9 to both Gly4-Pro7 and Ser6-Arg9, and of Phe5-Arg9 to Ser6-Arg9, Phe8-Arg9, and Ser6-Pro7, by unidentified peptidases. 7. A model for the enzymatic inactivation of bradykinin by rat brain neuronal perikarya is proposed.     

Cyclic enkephalin-deltorphin hybrids containing a carbonyl bridge: structure and opioid activity

Six hybrid N-ureidoethylamides of octapeptides in which an N-terminal cyclic structure related to enkephalin was elongated by a C-terminal fragment of deltorphin were synthesized on MBHA resin. The synthetic procedure involved deprotection of Boc groups with HCl/dioxane and cleavage of the peptide resin with 45 % TFA in DCM. d-Lys and d-Orn were incorporated in position 2, and Lys, Orn, Dab, or Dap in position 5. The side chains of the dibasic amino function were protected with the Fmoc group. This protection was removed by treatment with 55 % piperidine in DMF, and cyclization was achieved by treatment with bis-(4-nitrophenyl)carbonate. Using various combinations of dibasic amino acids, peptides containing a 17-, 18-, 19- or 20-membered ring structure were obtained. The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. Diverse opioid activities were observed, depending on the size of the ring. Extension of the enkephalin sequence at the C-terminus by a deltorphin fragment resulted in a change of receptor selectivity in favor of the δ receptor. The conformational propensities of selected peptides were determined using the EDMC method in conjunction with data derived from NMR experiments carried out in water. This approach allowed proper examination of the dynamical behavior of these small peptides. The results were compared with those obtained earlier with corresponding N-(ureidoethyl)pentapeptide amides.     

Limiting racemization and aspartimide formation in microwave-enhanced Fmoc solid phase peptide synthesis.

Microwave energy represents an efficient manner to accelerate both the deprotection and coupling reactions in 9-fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS). Typical SPPS side reactions including racemization and aspartimide formation can occur with microwave energy but can easily be controlled by routine use of optimized methods. Cysteine, histidine, and aspartic acid were susceptible to racemization during microwave SPPS of a model 20mer peptide containing all 20 natural amino acids. Lowering the microwave coupling temperature from 80 degrees C to 50 degrees C limited racemization of histidine and cysteine. Additionally, coupling of both histidine and cysteine can be performed conventionally while the rest of the peptide is synthesized using microwave without any deleterious effect, as racemization during the coupling reaction was limited to the activated ester state of the amino acids up to 80 degrees C. Use of the hindered amine, collidine, in the coupling reaction also minimized formation of D-cysteine. Aspartimide formation and subsequent racemization of aspartic acid was reduced by the addition of HOBt to the deprotection solution and/or use of piperazine in place of piperidine.     

Insect neuropeptide antagonist. Part II. Synthesis and biological activity of backbone cyclic and precyclic PBAN antagonists.

A new approach for the design and synthesis of pheromone biosynthesis activating neuropeptide (PBAN) agonists and antagonists using the backbone cyclization and cycloscan concepts is described. Two backbone cyclic (BBC) libraries were synthesized: library I (Ser library) was based on the active C-terminal hexapeptide sequence Tyr-Phe-Ser-Pro-Arg-Leu-NH2 of PBAN1-33NH2; whereas library II (D-Phe library) was based on the sequence of the PBAN lead linear antagonist Arg-Tyr-Phe-d-Phe-Pro-Arg-Leu-NH2. In both libraries the Pro residue was replaced by the BBC building unit Nalpha-(omega-aminoalkyl) Gly having various lengths of alkyl chain. The peptides of the two libraries were tested for agonistic and antagonistic activity. Four precyclic peptides based on two of the BBC antagonists were also synthesized; their activity revealed that a negative charge at the N-terminus of the peptide abolished antagonistic activity. We also describe the use of the reagent SiCl3I for selective deprotection of the Boc group from the building unit prior to on-resin amino-end to backbone-nitrogen (AE-BN) cyclization, during solid-phase synthesis with Fmoc chemistry.     

Chemistry of alpha-hydroxymethylserine: problems and solutions

Further improvements related to the synthesis of peptides containing HmS are presented. Efficient synthetic protocols have been developed to synthesize "difficult" sequences containing a C-terminal HmS residue, MeA-HmS or consecutive HmS. Preparative methods for orthogonal N- and/or C-protected HmS(Ipr) derivatives are described. Their compatibility with standard solution or solid-phase peptide chemistry protocols allows synthetic flexibility toward HmS-containing peptides. In the synthesis of the sterically hindered dipeptides with the C-terminal HmS(Ipr) residue, HATU proves the highest efficiency, as compared with the fluoride and PyBroP/DMAP coupling methods. The HATU method also outperforms the fluoride activation in the solid-phase assembly of HmS homosequence. Specific protocols are described to overcome an undesired cyclization to diketopiperazines that occurs during the removal of Fmoc from dipeptides with the C-terminal HmS(Ipr) or HmS residues, thus precluding their C-->N elongation. The successful protocols involve: (i) the 2+1 condensation using mixed anhydride activation yielding the desired product with the highest optical integrity or (ii) use of the 2-chlorotrityl resin as a solid support sterically suppressing the undesired cleavage due to diketopiperazine formation. The latter approach allows the mild conditions of peptide cleavage from solid support, preserving the isopropylidene protection and minimizing the undesired N-->O-acyl migration that was observed under prolonged acid treatment used for cleaving the HmS peptide from the Wang resin.     

Mass spectrometry analysis for the determination of side reactions for cyclic peptides prepared from an Fmoc/t}Bu/Dmab protecting group strategy

The Association of Biomolecular Resource Facilities (ABRF) Peptide Synthesis Research Group (PSRG) proposed for their annual study that laboratory members prepare cyclo(Tyr-Glu-Ala-Ala-Arg-DPhe-Pro-Glu-Asp-Asn) according to the following synthetic pathway: (i) side-chain anchoring Fmoc-Asp(OH)-ODmab to a Rink amide resin; (ii) linear assembly; (iii) Dmab and Fmoc removal, respectively; (iv) on-resin cyclization with an uronium-based coupling reagent; (v) final cleavage/deprotection with TFA. Based upon this protocol, a variety of side-products were identified:(i) N-terminal guanidine formation; (ii) C-terminal piperidyl amide formation; and (iii) a novel C-terminal benzyl amide-guanidine derivative that formed due to a chemical reaction between the Dmab protecting group and the uronium-based coupling agent. The elemental composition and subsequent structure determination of this unexpected derivative was established by tandem mass spectrometry, i.e. low energy collision-induced dissociation experiments with fragment mass determination within 5 ppm.     

Degradation of neurotensin by rabbit brain endo-oligopeptidase A and endo-oligopeptidase B (proline-endopeptidase)

The degradation of neurotensin and D-Tyr11 neurotensin by apparently homogeneous preparations of rabbit brain endo-oligopeptidase A and endo-oligopeptidase B (Proline-endopeptidase) was studied. Peptide fragments were isolated by high performance liquid chromatography and identified by amino acid analysis. Endo-oligopeptidase A cleaved neurotensin at the Arg8-Arg9 bond whereas D-Tyr11 neurotensin was not significantly hydrolysed. Endo-oligopeptidase B cleaved at the carboxyl side of Pro7, Pro10 in neurotensin and at Pro7 in D-Tyr11 neurotensin. The concentration dependent inhibition of neurotensin degradation by bradykinin and vice-versa represents additional evidence that endo-oligopeptidase A cleaves both Phe5-Ser6 bond of bradykinin and the Arg8-Arg9 bond of neurotensin.     

Mass spectrometry analysis for the determination of side reactions for cyclic peptides prepared from an Fmoc/t}Bu/Dmab protecting group strategy

Summary The Association of Biomolecular Resource Facilities (ABRF) Peptide Synthesis Research Group (PSRG) proposed for their annual study that laboratory members preparecyclo(Tyr-Glu-Ala-Ala-Arg-DPhe-Pro-Glu-Asp-Asn) according to the following synthetic pathway: (i) side-chain anchoring Fmoc-Asp(OH)-ODmab to a Rink amide resin; (ii) linear assembly; (iii) Dmab and Fmoc removal, respectively; (iv) on-resin cyclization with an uronium-based coupling reagent; (v) final cleavage/deprotection with TFA. Based upon this protocol, a variety of side-products were identified: (i)N-terminal guanidine formation; (ii)C-terminal piperidyl amide formation; and (iii) a novelC-terminal benzyl amide-guanidine derivative that formed due to a chemical reaction between the Dmab protecting group and the uronium-based coupling agent. The elemental composition and subsequent structure determination of this unexpected derivative was established by tandem mass spectrometry, i.e. low energy collision-induced dissociation experiments with fragment mass determination within 5 ppm.     

Peptides Derived from α-hydroxymethylserine: Aspects of solid-phase synthesis

Summary The solid-phase synthesis of peptides derived from the sterically hindered α-hydroxymethylserine (HmS) was investigated. The acid-sensitive,O,O-isopropylidene (Ipr) protection of HmS is compatible with the Fmoc chemistry, represented here by the Fmoc-HmS(Ipr)-OH and Fmoc-HmS(Ipr)-F derivatives. Three analogs of the opioid pentapeptide DADLE with a single or two consecutive HmS residue(s) were synthesized using Wang resin as the solid support. The HATU method has been shown to effectively accomplish ‘difficult’ couplings with the HmS(Ipr) residue. Wang resin is not suitable, for the synthesis of sequences with a C-terminal HmS because of the easy formation of the diketopiperazine resulting from the cyclization of the susceptible dipeptide sequence AA-HmS(Ipr) bound to the resin. A further drawback of the Wang resin methodology is the increased danger of the undersired N→O-acyl shift, when long-lasting acidic cleavage is applied. These side reactions are totally suppressed when the 2-chlorotrityl polystyrene is used as a solid support. The mild conditions (AcOH/TFE/DCM) applied for the peptide detachment from this resin do not affect the Ipr protection, affording highly pure fragments with HmS(Ipr) residues suitable for post-cleavage condensation, cyclization or controlled side-chain deprotection. This approach is documented by the efficient synthesis of linear and cyclic analogs of the opioid hexapeptide DTLET containing two residues of HmS or HmS(Ipr) in positions 2 and 6.     

Peptide-bond modification for metal coordination: peptides containing two hydroxamate groups

Peptide-bond modification via N-hydroxylation has been explored as a strategy for metal coordination to induce conformational rigidity and orient side chains for specific molecular recognition. N-Hydroxyamides were prepared by reacting N-benzyloxyamino acid esters or amides with Fmoc-AA-Cl/AgCN (Fmoc: 9-fluorenylmethoxycarbonyl; AA: amino acid) in toluene or Fmoc-AA/HATU/DIEA in DMF (HATU: O-(7-azabenzotriazol-lyl)-1,1,3,3-tetramethyluronium hexafluorophosphate; DIEA: N,N-diisopropylethylamine; DMF: N,N-dimethylformamide), followed by deblocking of benzyl protecting groups. Novel linear and cyclic N,N'-dihydroxypeptides were efficiently assembled using Fmoc chemistry in solution and/or on a solid support. As screened by electrospray ionization-mass spectroscopy (ESI-MS), high iron-binding selectivity and affinity were attainable. Compounds having a spacer of two alpha-amino acids between the amino acids bearing the two hydroxamates, i.e., a spacer of 8 atoms, generated 1:1 iron complex species in the gas phase. Moreover, high performance liquid chromatography (HPLC), uv/vis, and (1)H-NMR analyses provided direct evidence for complex formations in solution. Significantly, the representative compound cyclo(Leu-Psi[CON(OH)]-Phe-Ala-Pro)(2) (P8) may serve as a robust metal-binding scaffold in construction of a metal-binding library for versatile metal-mediated molecular recognition.     

Novel deprotection method of Fmoc group under neutral hydrogenation conditions

Novel deprotection method of the Fmoc (9-flurorenylmethoxycarbonyl) protective group under Pd/C-catalyzed hydrogenation conditions at room temperature was developed. The addition of CH₃CN accelerated the deprotection of the Fmoc group, and also the application of H₂ pressure (3 atm) shows notable rate enhancement.     

N‐(ureidoethyl)amides of cyclic enkephalin analogs

Novel N‐(ureidoethyl)amides of cyclic enkephalin analogs have been synthesized. The p‐nitrophenyl carbamate of 1‐Boc‐1,2‐diaminoethane was coupled with 4‐methylbenzhydrylamine (MBHA) resin. The Boc group was removed by treatment with HCl/dioxane, and the peptide chain was assembled using Boc strategy. For deprotection of amino function, HCl/dioxane was used. D ‐Lys or D ‐Orn were incorporated in position 2, and the side chains of Lys, Orn, Dab, or Dap in position 5 were protected with Fmoc group. Side chain protection was removed by treatment with 55% piperidine in DMF, and cyclization was achieved by treatment with bis‐(4‐nitrophenyl)carbonate to form a urea bridge. The peptide was cleaved from the resin by treatment with 45% TFA in DCM. The peptides were tested in the guinea‐pig ileum (GPI) and mouse vas deferens (MVD) assays. Divers opioid activities were observed, depending on the size of the ring. In comparison with [Leu⁵]enkephalin, all peptides were more active in the GPI assay (between 125 and 12 times), and some of them were also more potent in the MVD assay. The conformational propensities of each peptide were determined using the EDMC method in conjunction with NMR experiments. This approach allows treating the dynamical behavior of small peptides properly. The results were compared with those obtained previously for corresponding nonsubstituted amides and are in agreement with the biologically active conformation proposed by us earlier. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Search for new synthetic immunosuppressants II. Tetrazole analogues of hymenistatin I

Linear and cyclic hymenistatin I (HS I) analogues with dipeptide segments Ile2-Pro3 Pro3-Pro4 and Val6-Pro7 replaced by their tetrazole analogues Ile2-psi[CN4]-Ala3', Pro3-psi[CN4]-Ala4 and Val6-psi[CN4]-Ala7 were synthesized by the solid phase peptide synthesis method and cyclized with the TBTU and/or HATU reagent. The peptides were examined for their immunosuppressive activity in the lymphocyte proliferation test (LPT).