Design of protecting groups for the β-carboxylic group of aspartic acid that minimize base-catalyzed aspartimide formation

With the objectives of developing new protecting groups for the β-carboxyl group of aspartic acid that are resistant to base-catalyzed aspartimide formation and of evaluating the importance of sterical factors in the design of such protecting groups, four new alkyl ester derivatives of aspartic acid were synthesized. The β-3-pentyl, β-4-heptyl, β-2,6-dimethyl-4-heptyl and the recently described β-2,4-dimethyl-3-pentyl esters of Boc-aspartic acid were incorporated into model peptides, and the resin-bound protected peptides were treated with 20% pipetidine for 10 h. The levels of aspartimide-related side products were compared with the previously reported β-cyclohexyl, β-menthyl and β-2-adamantyl esters of aspartic acid. The results show that bulky, acyclic, aliphatic protecting groups (in particular the 2,4-dimethyl-3-pentyl ester) are significantly more resistant to base-catalyzed aspartimide formation than comparably rigid cyclic alkyl esters that under the same reaction conditions form several-fold more aspartimide-related side products. Using elevated temperatures to overcome difficult couplings leads to the formation of significant amounts of aspartimide when aspartic acid is protected with the cyclohexyl group, but the 2,4-dimethyl-3-pentyl protecting group offers excellent protection under these conditions. The use of the 2,4-dimethyl-3-pentyl protecting group will allow the use of orthogonally removable base-labile protecting groups in Boc chemistry and suggests a design of protecting groups for other nucleophile-sensitive trifunctional amino acids in both Boc and Fmoc chemistry. © Munksgaard 1996.     

Design and synthesis of sulfur-free cyclic hexapeptides which contain the RGD sequence and bind to the fibrinogen GP IIb/IIIa receptor

The Arg-Gly-Asp (RGD) sequence is the key recognition site in many adhesive interactions. To probe the structural and conformational requirements for potential antithrombotic agents, we have designed and synthesized three cyclic hexapeptides ( 1, 5 and 6 ) containing the RGD sequence. In the ELISA GP IIb/IIIafibrinogen receptor assay, 1, 5 and 6 bound with IC₅₀ values of 1, 0.1 and 0.016 μm, respectively. All three peptides completely displaced fibrinogen from the receptor. No potent, sulfur-free cyclic hexapeptide had heretofore been described as a fibrinogen receptor antagonist. The enhanced binding affinity of 6 , distinguished by the presence of two d -amino acids, is likely to reflect an increased conformational resemblance to the natural peptide ligands. Cyclization of H-Asp(OFm)-d Ser-Phe-d Phe-Arg-Gly-OH with DPPA and NaHCO₃ in DMF to afford 6 was attended by subsequent aspartimide formation with generation of 9-fluorenylmethanol. Interestingly, imide formation was not observed with any of the three linear hexapeptides ( 3, 8 and 9 ), with the all-l -cyclic peptide 1 , nor with 5 , which contains only Ser-1 in the D-configuration. The observed imide formation led us to use catalytic transfer hydrogenation rather than piperidine to remove the 9-fluorenylmethyl ester protecting group at the β-carbonyl of aspartic acid. Further investigation revealed that imide formation was minimized by careful exclusion of water, reducing dissolution of NaHCO₃. Thus the distinguishing conformational features of 6 express themselves both in receptor affinity and chemical propensity toward imide formation.     

Inhibition of the respiratory-linked membrane potential in E. coli membrane vesicles by octapeptin.

Octapeptin is a peptide antibiotic which affects bacterial membrane structure and selective membrane permeability for protons and potassium. The influence of octapeptin on the formation of a membrane potential generated across bacterial vesicles was monitored using the Rb+-valinomycin transport system. Octapeptin inhibited the respiratory-linked generation of membrane potentials formed in the presence of succinate or Asc/PMS. In addition, the antibiotic inhibited [3H]-leucine transport driven either by succinate or Asc/PMS. These studies support the proposal that the antimicrobial activity of octapeptin is due to inhibition of the formation of a membrane potential generated in the presence of appropriate respiratory substrates.     

Application and comparison of two-rate and differential kinetic methods in simultaneous spectrophotometric determination of ascorbic acid and L-cysteine in pharmaceutical samples

Two methods for the simultaneous determination of ascorbic acid (Asc) and L-cysteine (Cys) from kinetic spectrophotometric data are described and compared. The first is the two-rate method (TRM), and the second, the differential kinetic method (DKM). Present in an excess amount, iron(III) is quantitatively reduced by Asc or Cys to iron(II) that, in turn, interacts with phenanthroline (Phen) to form a colored product, representing a Fe(II)-Phen complex that possesses a maximum absorbance at λ_max = 510 nm. The first method is based on the measurement of reaction rates at two points in the course of successive reactions, and the second method is based on the difference in kinetics of the Asc and Cys oxidation reactions at different pH values. Both oxidation reactions have been studied by fitting the kinetic curves (absorbance versus time) to suitable reaction rate equations. It is established that the Cys + Fe(III) reaction obeys simple first-order kinetics, while the Asc + Fe(III) is a successive two-step process. Under the conditions studied, both Asc and Cys are determined in the 0.5–5.0 ppm range with satisfactory accuracy. The two methods under consideration have been successfully used for the analysis of model and real samples. The RMSEP values for the determination of Cys and Asc by the two kinetic methods (TRM, DKM) are 0.121, 0.120 and 0.131, 0.303 and the corresponding REP values are 5.15, 5.12% and 5.18, 11.98%, respectively. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 40, No. 1, pp. 41–48, January, 2006.     

The effects of a histidine residue on the C-terminal side of an asparaginyl residue on the rate of deamidation using model pentapeptides

The effects of a histidine (His) residue located on the C-terminal side of an asparaginyl (Asn) residue on the rate of deamidation were studied using Gly-Gln-Asn-X-His pentapeptides. The rates of deamidation of the pentapeptides were determined at 37 degrees C (I = 0.5) as function of pH, buffer species, and buffer concentration. A capillary electrophoresis stability-indicating assay was developed to monitor simultaneously the disappearance of the starting peptides and the appearance of the degradation products. The rates of degradation of the peptides were pH dependent, increasing with pH, and followed apparent first-order kinetics. At pH values <6.5, Gly-Gln-Asn-His-His degraded faster than Gly-Gln-Asn-Gly-His, suggesting that the N+1 His residue is catalyzing the deamidation of the Asn residue. The His side chain at these pH values could function as a general acid catalyst, stabilizing the oxyanionic transition state of the cyclic imide intermediate formation. In contrast, at pH values >6.5, Gly-Gln-Asn-Gly-His deamidates more rapidly than Gly-Gln-Asn-His-His. The bulk of the side chain of the N+1 His residue versus the N+1 Gly residue apparently inhibits the flexibility of the peptide around the reaction site and, consequently, reduces the rate of the reaction. The significance of this steric hindrance effect of the N+1 His residue on the rate of deamidation was examined further. It was observed that at pH >6.0, Gly-Gln-Asn-His-His undergoes deamidation faster than Gly-Gln-Asn-Val-His. This observation indicated that, at the higher pH values, the N+1 His residue is also acting as a catalyst. Thus, at basic pH, the N+1 His residue influences the rate of deamidation via two opposing effects; that is, general base catalysis and steric interference. The pentapeptide Gly-Gln-Asn-His-His, in addition to undergoing the deamidation reaction, also undergoes bond cleavage at the Asn-His peptide bond. The enhanced rate of Asn-His peptide bond cleavage can be attributed to the general base behavior of the His residue, leading to increased nucleophilicity of the Asn side-chain amide group. Finally, we have shown that the His residue that is two amino acids removed from the Asn, the N+2 position, has little or no effect on the rate of deamidation.     

Esterification of 7-theophyllineacetic acid with diethylene glycol monomethyl ether

The kinetics of esterification of 7-theophyllineacetic acid with diethylene glycol monomethyl ether in the presence of dicyclohexylcarbodiimide and 4-dimethylaminopyridine as catalyst was studied. According to the known mechanism, besides the main process, the side-reaction of intramolecular rearrangement with formation of pharmacologically active N-acylurea occurs. The course of the main and the side-process was monitored by RP-HPLC with UV-detection. For that purpose, quantification of both ester and N-acylurea in the reaction mixture was performed. Influence of the concentration of the reactants (acid, alcohol and catalyst) on the progress of esterification and preparation of the by-product was investigated. Based on the obtained results, the reaction conditions leading to maximal yield of the ester and N-acylurea are proposed. The possibility of turning esterification to the synthesis of the side-product was also found. Reactions of the preparation of both the ester and N-acylurea were found to follow first-order kinetics. The rate constants of both processes were estimated.     

Utilization of 1-hydroxybenzotriazole in mixed anhydride coupling reactions

The coupling of Boc-Val-OH to either H-Pro-OBzl or H-Pro-Gly-Val-Gly-OBzl by the mixed anhydride method leads to the formation of a urethane by-product in yields of 40–60%. This side reaction can be suppressed by the addition of HOBt to the reaction mixture before the amino component is added. This results in a substantially increased yield of the desired peptide.     

Amino acids and peptides. XVIII. Dipeptide formation during the synthesis of Z-Asp(OBzl)-OH

During the benzyloxycarbonylation of H-Asp(OBzl)-OH by the Schotten-Bauman reaction with benzyloxycarbonyl chloride in the presence of NaHCO₃ or Na₂CO₃, besides Z-Asp(OBzl)-OH, Z-Asp(OBzl)-Asp(OBzl)-OH was formed as side product, although the extent of the dipeptide formation differed depending on the base used (10% and 20% respectively). It was found that melting point, rotation value and Rf values upon thin-layer chromatography of Z-Asp(OBzl)-Asp(OBzl)-OH were quite similar to those of Z-Asp(OBzl)-OH.     

SIDE REACTIONS IN PEPTIDE SYNTHESIS:

Acylation of amino acid β-naphthylamides with protected (Boc) amino acid-isobutylcarbonic acid mixed anhydrides resulted in each case in the formation of some undesired by-product: an isobutyloxycarbonylamino acid β-naphthylamide. The amount of this second acylation product was particularly high, with the hindered amino acids valine and isoleucine as carboxyl-components. The nature of the amino component had no major influence on the extent of this side reaction.     

Solid phase synthesis of the protected 43–55 tridecapeptide of the heavy chain of myeloma immunoglobin M603, employing cyclohexyl ester protection for glutamic acid

A protected tridecapeptide of the sequence Boc-Lys(2ClZ)-Arg(Tos)-Leu-Glu(OcHex)-Trp(For)-Ile-Ala-Ala-Ser(Bzl)-Arg(Tos)-Asn-Lys(2ClZ)-Gly-OH, representing residues 43–55 of the variable region of the heavy chain of mouse myeloma protein M603, was synthesized. It was assembled by a stepwise solid phase method designed to give a fully protected peptide in high yield and purity with minimal side reactions. Thus, the peptide chain was attached as an α-methyl phenacyl ester to a 2-bromopropionyl-resin. After the synthesis the protected peptide fragment was obtained in 89% yield by photolytic cleavage from the resin. The peptide was purified by multiple precipitation and column chromatography. It was shown to be homogeneous by reverse phase high pressure liquid chromatography, and it had the correct amino acid composition and sequence. In the course of this work it was shown that tert.-butyloxycarbonyl-amino acids caused the formation of significant amounts of pyrrolidone carboxylic acid residues during the coupling reaction when a γ-benzyl glutamyl residue was NH₂-terminal. Other weak-acid additives also caused this chain terminating side reaction. The cyclization was markedly suppressed by protection of the glutamyl side chain as a cyclohexyl ester. With this protecting group, no evidence of pyrrolidone carboxylic acid formation could be detected in the tridecapeptide 43–55.     

长效避孕药——17β-甾醇酯的合成

本文报道了为寻找长效避孕药而设计的炔诺酮酯和18-甲基炔诺酮酯的合成方法。以双环己基碳二亚胺(DCC)为脱水剂,4-二甲氨基吡啶(DMAP)为接触剂,酸与醇直接反应,合成了五个17β-酯,收率为60～90%。     

Myocardial salvage by trolox and ascorbic acid,but not ascorbic acid alone,in anesthetized dogs and rabbits

The myocardial salvaging properties of Trolox (Trix; a water- and lipid-soluble vitamin E analog with antioxidant properties) and ascorbic acid (Asc; a water-soluble antioxidant) were evaluated in anesthetized male dogs and rabbits. Myocardial infarction (MI) was induced by occlusion and reperfusion of the left anterior descending coronary artery: a 2-hr occlusion and 4-hr reperfusion in dogs, and a 15-min occlusion and 3-hr reperfusion in rabbits. This occlusion/reperfusion protocol induced %MI (MI normalized to area at risk) of approximately 20% beyond that induced by occlusion alone in both species. Trlx, Asc (100 and 150 mg/kg/injection, respectively, by injection; or 100 and 150 mg/kg/min, respectively, by continuous infusion), or vehicle (Veh) were administered into the ascending aorta in dogs and into the left atrium in rabbits. In severely ischemic dogs (myocardial collateral blood flow < 0.1 ml/min/g), a single injection of Trlx plus Asc, but not Asc alone, at the onset of reperfusion reduced %MI (Trlx + Asc = 24.3 ± 5.4%, n = 7; Asc = 50.1 ± 9.6%, n = 4; Veh = 45.0 ± 5.0%, n = 5). In rabbits, %MI was reduced (or tended to be reduced) by either 3 hourly injections (Trlx + Asc = 33.3 ± 4.0%, n = 19; Asc = 48.3 ± 6.3%, n = 11; Veh = 43.7 ± 3.3%, n = 8) or an injection followed by continuous infusion (Trlx + Asc = 36.0 ± 4.0%, n = 9; Veh = 49.0 ± 3.0%, n = 8), beginning at the onset of reperfusion. %MI in rabbits was not affected by either hourly injections beginning from 1 hr after, or a single injection at 1–3 × the dosages (i.e., 100–300 and 150–450 mg/kg for Trlx and Asc, respectively), during onset of reperfusion. Hemodynamic variables (arterial pressure, heartrate, rate-pressure-product and left ventricular contractility) were similar among all treat- ment groups in both species. In conclusion, Trlx/Asc combination, but not Asc alone, re- duces %MI in dogs and rabbits. The salvaging effects of Trlx/Asc are unrelated to any changes in hemodynamics. These results are consistent with the hypothesis that lipid peroxidation is involved in myocardial reperfusion injury. © 1992 Wiley-Liss, Inc.     

二环杂环化合物热重排反应的意外副产物7-甲硫基-4-甲基-2H-色烯-2-酮及其抗HBV活性

目的 利用7-二甲基硫代氨甲酰氧基-4-甲基-2H-色烯-2-酮(3)高温下的热重排反应制备7-二甲基氨甲酰基硫基-4-甲基-2H-色烯-2-酮(5).方法 热重排反应.结果 除了得到期望的7-二甲基氨甲酰基硫基-4-甲基-2H-色烯-2-酮(5)产物外,还分离得到意外的副产物7-甲硫基-4-甲基-2H-色烯-2-酮(7),其结构经波谱确证.对该副产物进行抗-HBV生物活性测定,显示出显著的抗HBV活性.结论 7-二甲基硫代氨甲酰氧基-4-甲基-2H-色烯-2-酮(3)在高温下产生7-甲硫基-4-甲基-2H-色烯-2-酮(7)副产物,未见文献报道,并对其产生的机制进行探讨.首次报道了7-甲硫基-4-甲基-2H-色烯-2-酮(7)的抗HBV活性.     

Substitution on the Phe3 aromatic ring in cyclic delta opioid receptor-selective dermorphin/deltorphin tetrapeptide analogues: electronic and lipophilic requirements for receptor affinity.

In an effort to explore structural features affecting receptor recognition in a series of conformationally restricted tetrapeptides related to the cyclic, delta opioid receptor-selective analogue, [formula: see text] electronic, lipophilic, and steric effects at the Phe3 residue were assessed by substitution at different positions of the side-chain aromatic ring by halogens, alkyl, hydroxyl, and nitro groups. Effects on opioid receptor binding affinity and selectivity were determined. The results, which are generally consistent with reports of analogous modifications in linear and cyclic pentapeptide enkephalins, indicate that steric, lipophilic, and electronic properties are all important determinants of delta opioid receptor recognition. Specifically, modifications which increase lipophilicity or exert electron-withdrawing effects on the aromatic ring enhance binding affinity, while hydrophilic, bulky, or electron-releasing modifications are detrimental. These observations are in excellent agreement with quantitative structure-activity relationship (QSAR) results reported for Phe4 modifications in linear opioid pentapeptide enkephalin analogues, suggesting that the Phe3 tetrapeptide side chain and the Phe4 pentapeptide side chain interact with the same delta receptor binding subsite.     

Ca2+-mediated ascorbate release from coronary artery endothelial cells

1.--The addition of Ca(2+) ionophore A23187 or ATP to freshly isolated or cultured pig coronary artery endothelial cells (PCEC) potentiated the release of ascorbate (Asc). Cultured PCEC were used to characterize the Ca(2+)-mediated release. An increase in Ca(2+)-mediated Asc release was observed from PCEC preincubated with Asc, Asc-2-phosphate or dehydroascorbic acid (DHAA). 2.--The effects of various ATP analogs and inhibition by suramin were consistent with the ATP-induced release being mediated by P2Y2-like receptors. 3.--ATP-stimulated Asc release was Ca(2+)-mediated because (a) ATP analogs that increased Asc release also elevated cytosolic [Ca(2+)], (b) Ca(2+) ionophore A23187 and cyclopiazonic acid stimulated the Asc release, (c) removing extracellular Ca(2+) and chelating intracellular Ca(2+)inhibited the ATP-induced release, and (d) inositol-selective phospholipase C inhibitor U73122 also inhibited this release. 4.--Accumulation of Asc by PCEC was examined at Asc concentrations of 10 microM (Na(+)-Asc symporter not saturated) and 5 mM (Na(+)-Asc symporter saturated). At 10 microM Asc, A23187 and ATP caused an inhibition of Asc accumulation but at 5 mM Asc, both the agents caused a stimulation. Substituting gluconate for chloride did not affect the basal Asc uptake but it abolished the effects of A23187. 5.--PCEC but not pig coronary artery smooth muscle cells show a Ca(2+)- mediated Asc release pathway that may be activated by agents such as ATP.     

FORMATION OF AMINOSUCCINYL PEPTIDES DURING ACIDOLYTIC DEPROTECTION FOLLOWED BY THEIR TRANSFORMATION TO PIPERAZINE-2, 5-DIONE DERIVATIVES IN NEUTRAL MEDIA

Prolonged acidic treatment of Boc-Leu-Asp(OBu^t)-Phe-NH₂ with 4N HCl in acetic acid resulted in H-[Leu-Asc-Phe-NH₂] HCl(Asc, aminosuccinyl), which transformed partially to cyclo[Leu-Asp(Phe-NH₂)] during its purification by column chromatography on silica gel with a mixture of ethyl acetate/pyridine/acetic acid/water = 60:20:6:11, i.e. in neutral medium. Examination of the imide formation was extended to different reaction conditions (no imide derivative was detected in trifluoroacetic acid), to several protected derivatives of L-aspartyl-L-phenylalaninamide and to tripeptides containing an aspartyl residue in the middle position. It was clearly demonstrated that in strongly acidic media the imide derivatives are directly formed from the aspartyl peptides containing a free β-carboxyl group. The influence of the C-terminal residue was greater than the N-terminal on both the rate of formation of the imide and its further transformation to piperazine-2,5-dione derivative. In aqueous ethanol the X-Asc-Y-NH₂ (X, Pro, Leu; Y, Gly, Ala, Val, Phg, Phe) containing N-terminal proline are more readily transformed to piperazine-2,5-dione derivatives, but compared to simple proline dipeptides the rate of this transformation is relatively slow because of the crowdedness of the tricyclic transitional state.     

Ring formation in a pentapeptide with alternating L and D residues: an analogy to cyclization in the biosynthesis of peptide antibiotics

Acetylation of L-isoleucyl-D-alanyl-D-alanyl-L-valyl-D-leucine with acetic anhydride followed by methylation with diazomethane yielded the expected acetylpentapeptide methyl ester with molecular weight 541, but also resulted in the formation of a by-product with molecular weight 555. The incorporation of the mass corresponding to CH2 seems to be due to ring closure--via a mixed anhydride--and methylation of the cyclol derivative thus formed. A preferred, ring-like conformation stabilized by intramolecular hydrogen bonds that in turn are the consequences of the alternation of D- and L- residues in the sequence, is invoked as explanation for the unexpected cyclization. This assumption is supported by the conversion of the pentapeptide methyl ester to desthiomalformain in molten imidazole.     

Effect of adjacent histidine and cysteine residues on the spontaneous degradation of asparaginyl- and aspartyl-containing peptides

Aspartate and asparagine residues in polypeptides are subject to nonenzymatic reactions that lead to deamidation, isomerization, peptide bond cleavage and racemization. Much of this reactivity is due to the propensity for the initial formation of a cyclic succinimide intermediate. We have been interested in determining the effect of the side chains of neighboring histidine and cysteine residues in facilitating these reactions, particularly in the possibility that they can act as general acids and bases. In this study, we found little or no effect of histidine residues preceding an asparagine residue in hexapeptides derived from the sequence of adrenocorticotropic hormone, while a histidine residue preceding an aspartic acid residue was found to increase the rate of succinimide formation 8- to 11-fold. The presence of a histidine residue following either an asparagine or aspartic acid residue did not effect the rate of succinimide formation by peptide-bond nitrogen attack, but did increase the rate of the competing side-chain nitrogen attack leading to cleavage in the asparaginyl-containing peptide. We found that the effect of a cysteine residue following an asparagine or aspartic acid residue was in general similar to that of a serine residue, although the cleavage reaction appeared to be enhanced. These results suggest that His-Asp sequences may be particularly labile to spontaneous degradation in proteins and peptides, possibly owing to the ability of the histidine residue to facilitate succinimide formation by protonating the OH^? leaving group on the side chain carboxylic acid of the aspartic acid residue. Finally, we have also utilized these results, along with previously accumulated data on succinimide formation in related peptides, to correlate the rate of succinimide formation with the predicted acidity of the peptide bond nitrogen atom that is involved in the initial nucleophilic attack. © Munksgaard 1995.     

Mechanism of 1,N2-etheno-2'-deoxyguanosine formation from epoxyaldehydes

Background levels of etheno adducts have been attributed to the reaction of DNA with 2,3-epoxyaldehydes, a proposed product of lipid peroxidation. We have examined the reaction of (2R,3S)-epoxyhexanal with dGuo to give 7-(1S-hydroxybutyl)-1,N(2)-etheno-dGuo. We observed that the stereochemistry of the side chain scrambled over time. This process provided insight into the mechanism for the formation of 1,N(2)-etheno-dGuo from 4,5-epoxy-2-decenal [Lee, S. H., et al.(2002) Chem. Res. Toxicol. 15, 300-304]. The mechanistic proposal predicts that 2-octenal is a by-product of the reaction. The reaction of 4,5-epoxy-2-decenal was reinvestigated, and the 2-octenal adduct of dGuo was identified as a product of this reaction in support of the mechanistic proposal. Also observed are products that appear to be derived from 2,3-epoxyoctanal, which can be formed through Schiff base formation of 4,5-epoxy-2-decenal with the dGuo followed by hydration of the double bond and retro-aldol reaction.