Epimerization in peptide thioester condensation

Peptide segment couplings are now widely utilized in protein chemical synthesis. One of the key structures for the strategy is the peptide thioester. Peptide thioester condensation, in which a C‐terminal peptide thioester is selectively activated by silver ions then condensed with an amino component, is a powerful tool. But the amino acid adjacent to the thioester is at risk of epimerization. During the preparation of peptide thioesters by the Boc solid‐phase method, no substantial epimerization of the C‐terminal amino acid was detected. Epimerization was, however, observed during a thioester–thiol exchange reaction and segment condensation in DMSO in the presence of a base. In contrast, thioester–thiol exchange reactions in aqueous solutions gave no epimerization. The epimerization during segment condensation was significantly suppressed with a less polar solvent that is applicable to segments in thioester peptide condensation. These results were applied to a longer peptide thioester condensation. The epimer content of the coupling product of 89 residues was reduced from 27% to 6% in a condensation between segments of 45 and 44 residues for the thioester and the amino component, respectively. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Suppression of epimerization by cupric (II) salts in peptide synthesis using free amino acids as amino components.

An epimerization-free system for coupling N-protected peptides with free amino acids was developed. A number of inorganic substances were tested as epimerization suppressant additives during the coupling by various methods (carbodiimide plus additives, uronium salts, Woodward's reagent-K, isobutyl-chloroformate, etc.). Some of them (ZnCl2, RbClO4, LiCl, SnCl4, AlCl3, etc.) in combination with some coupling methods can guarantee coupling with minimal epimerization (D-epimer < 1%). But only a simultaneous use of 1-hydroxybenzotriazole and Cu2+ ions as additives in carbodiimide-mediated peptide couplings appeared to give a standard result (D-epimer < 0.1%). There was no epimerization even in the case when N-methyl amino acid (sarcosine) was used as an amino component, while in the absence of Cu2+ ions an unacceptable level of epimerization was observed (D-epimer, 22% for carbodiimide with the 1-hydroxybenzotriazole method). So far it has been considered that Cu2+ ions prevent obtaining peptides in high yields (< 90%) by various coupling methods. We have found that the use of 1-hydroxybenzotriazole, CuCl2 and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide instead of dicyclohexylcarbodiimide provides a possible method for obtaining the desired peptides in 90-99% yields without epimerization. All these results were shown by employing several model peptide couplings with free amino acids as amino components dissolved in an effective solvent system which readily dissolved them.     

Tunable degradation of maleimide-thiol adducts in reducing environments

Addition chemistries are widely used in preparing biological conjugates, and in particular, maleimide-thiol adducts have been widely employed. Here, we show that the resulting succinimide thioether formed by the Michael-type addition of thiols to N-ethylmaleimide (NEM), generally accepted as stable, undergoes retro and exchange reactions in the presence of other thiol compounds at physiological pH and temperature, offering a novel strategy for controlled release. Model studies ((1)H NMR, HPLC) of NEM conjugated to 4-mercaptophenylacetic acid (MPA), N-acetylcysteine, or 3-mercaptopropionic acid (MP) incubated with glutathione showed half-lives of conversion from 20 to 80 h, with extents of conversion from 20% to 90% for MPA and N-acetylcysteine conjugates. After ring-opening, the resultant succinimide thioether did not show retro and exchange reactions. The kinetics of the retro reactions and extent of exchange can be modulated by the Michael donor's reactivity; therefore, the degradation of maleimide-thiol adducts could be tuned for controlled release of drugs or degradation of materials at time scales different than those currently possible via disulfide-mediated release. Such approaches may find a new niche for controlled release in reducing environments relevant in chemotherapy and subcellular trafficking.     

Preparation of peptide-protein immunogens using N-succinimidyl bromoacetate as a heterobifunctional crosslinking reagent

Synthetic peptides derived from human fibrin were unidirectionally conjugated to three carrier proteins (bovine serum albumin, bovine alpha-lactalbumin, and keyhole limpet hemocyanin) by a method that employs N-succinimidyl bromoacetate. This heterobifunctional crosslinking reagent was prepared with a 79% yield in gram quantities from inexpensive starting materials. With this reagent, carrier proteins were first bromoacetylated, then reacted with the thiol groups of cysteine-containing peptides. The extent of peptide conjugation was assessed by amino acid analysis after acid hydrolysis, which liberated 1 mol of S-carboxymethylcysteine for each mole of thioether linkage between peptide and protein. The results of several conjugation experiments indicated that the efficiency of peptide incorporation ranged between 22 and 37% based on the recovery of S-carboxymethylcysteine relative to lysine. When the conjugates were used as immunogens, the S-carboxymethyl linkage was not antigenic in comparison with the S-maleimidobenzoyl linkage, even though their antipeptide immunoreactivities were similar.     

Stabilisation of a short α‐helical VIP fragment by side chain to side chain cyclisation: a comparison of common cyclisation motifs by circular dichroism

A model octapeptide segment derived from vasoactive intestinal peptide (VIP) was utilised to investigate the effect of several conventional cyclisation methods on the α‐helical conformation in short peptide fragments. Three of the classical macrocyclisation techniques (i.e. lactamisation, ring‐closing metathesis and Huisgen cycloaddition) were applied, and the conformations of the resulting cyclic peptides, as well as their linear precursors, were compared by CD analysis. The visibly higher folding propensity of the triazole‐tethered peptide after azide‐alkyne CuAAC macrocyclisation illustrates that the secondary structure of a short peptide fragment can differ significantly depending on the chemical strategy used to covalently cross‐link side chain residues in a ‘helical’ fragment. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Identification of cysteine as the reactive group in pyruvate kinase alkylated by 5-chloro-4-oxopentanoic acid.

4-Hydroxypentanoic acid alanine thioether was synthesized and characterized by n.m.r. spectroscopy. This derivative corresponded to the modified amino acid obtained by allowing 5-chloro-4-oxo[3,5-3H]pentanoic acid to react with rabbit muscle pyruvate kinase. Performic acid oxidation of 4-oxo[3,5-3H]pentanoic acid alanine thioether in pyruvate kinase gave [3H]succinate (67%) and [3H]carboxymethylcysteine (33%) as expected. Evidence is presented to show that NaBH4 reduction followed by periodate oxidation and analysis of radioactive formaldehyde production may provide a convenient method for distinguishing between thiol and amino alkylation by halogenomethyl ketone compounds. Peptide 'mapping' confirms that the modification by 5-chloro-4-oxopentanoic acid occurs primarily at one region of pyruvate kinase.     

A switchable stapled peptide

The O‐N acyl transfer reaction has gained significant popularity in peptide and medicinal chemistry. This reaction has been successfully applied to the synthesis of difficult sequence‐containing peptides, cyclic peptides, epimerization‐free fragment coupling and more recently, to switchable peptide polymers. Herein, we describe a related strategy to facilitate the synthesis and purification of a hydrophobic stapled peptide. The staple consists of a serine linked through an amide bond formed from its carboxylic acid function and the side chain amino group of diaminopropionic acid and through an ester bond formed from its amino group and the side chain carboxylic acid function of aspartic acid. The α‐amino group of serine was protonated during purification. Interestingly, when the peptide was placed at physiological pH, the free amino group initiated the O‐N shift reducing the staple length by one atom, leading to a more hydrophobic stapled peptide. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of N alpha-(tert-butoxycarbonyl)-N epsilon-[N-(bromoacetyl)-beta-alanyl]-L-lysine: its use in peptide synthesis for placing a bromoacetyl cross-linking function at any desired sequence position.

A new amino acid derivative, N alpha-(tert-butoxycarbonyl)-N epsilon-[N-(bromoacetyl)-beta-alanyl]-L-lysine (BBAL), has been synthesized as a reagent to be used in solid-phase peptide synthesis for introducing a side-chain bromoacetyl group at any desired position in a peptide sequence. The bromoacetyl group subsequently serves as a sulfhydryl-selective cross-linking function for the preparation of cyclic peptides, peptide conjugates, and polymers. BBAL is synthesized by condensation of N-bromoacetyl-beta-alanine with N alpha-Boc-L-lysine and is a white powder which is readily stored, weighed, and used with a peptide synthesizer, programmed for N alpha-Boc amino acid derivatives. BBAL residues are stable to final HF deprotection/cleavage. BBAL peptides can be directly coupled to other molecules or surfaces which possess free sulfhydryl groups by forming stable thioether linkages. Peptides containing both BBAL and cysteine residues can be self-coupled to produce either cyclic molecules or linear peptide polymers, also linked through thioether bonds. Products made with BBAL peptides may be characterized by amino acid analysis of acid hydrolyzates by quantification of beta-alanine, which separates from natural amino acids in suitable analytical systems. Where sulfhydryl groups on coupling partners arise from cysteine residues, S-(carboxymethyl)cysteine in acid hydrolyzates may also be assayed for this purpose. Examples are given of the use of BBAL in preparing peptide polymers and a peptide conjugate with bovine albumin to serve as immunogens or model vaccine components.     

Synthesis of peptide thioesters via an N–S acyl shift reaction under mild acidic conditions on an N‐4,5‐dimethoxy‐2‐mercaptobenzyl auxiliary group

An efficient method of peptide thioester synthesis is described. The reaction is based on an N‐4,5‐dimethoxy‐2‐mercaptobenzyl (Dmmb) auxiliary‐assisted N‐S acyl shift reaction after assembling a peptide chain by Fmoc‐solid phase peptide synthesis. The Dmmb‐assisted N‐S acyl shift reaction proceeded efficiently under mildly acidic conditions, and the peptide thioester was obtained by treating the resulting S‐peptide with sodium 2‐mercaptoethanesulfonate. No detectable epimerization of the amino acid residue adjacent to the thioester moiety in the case of Leu was found. The reactions were also amenable to the on‐resin preparation of peptide thioesters. The utility was demonstrated by the synthesis of a 41‐mer peptide thioester, a phosphorylated peptide thioester and a 33‐mer peptide thioester containing a trimethylated lysine residue. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Novel strategy for the synthesis of template-assembled analogues of rat relaxin.

The 'template-assembled synthetic protein' (TASP) concept provides a simple and elegant approach for the preparation of analogues that retain key structural elements. We have synthesized TASP molecules containing the putative active site of relaxin, a peptide that has similar structural features to insulin but a markedly different biological role. Two types of chemoselective thiol ligation strategies (thioether and thiazolidine) were used and compared. The synthetic pendant peptides contain an essential region for bioactivity that is located in the alpha-helical region of the relaxin B-chain. Depending on whether the thioether or the thiazolidine chemistry was used to attach the peptides to the template, the reacting amino acid was placed either at the C-terminus or N-terminus, respectively, thus allowing the choice of orientation relative to the carrier molecule. The template molecule consists of a decapeptide with two proline-glycine turns and four evenly spaced lysine residues that were functionalized with the appropriate chemical moiety. This allowed reaction with the appropriately derivatized peptides in solution. To improve the template ligation step using the thioether approach, a pendant peptide C-terminal cysteamine residue was used to reduce potential steric hindrance during conjugation. The design of the peptides as well as the synthetic strategy resulted in the acquisition of mimetics showing weak non-competitive and weak competitive antagonist properties.     

真养产碱杆菌112R4酰亚胺酶基因的克隆、序列分析及其在大肠杆菌中的表达

筛选到一株具海因水解活力的微生物,经鉴定后命名为真养产碱杆菌112R4。该菌能水解海因、二氢尿嘧啶和琥珀酰亚胺,且对琥珀酰亚胺活力最高,但不水解5单取代海因和5,5’双取代海因,因而被确定为含有酰亚胺酶。真养产碱杆菌112R4能在以琥珀酰亚胺为唯一碳、氮源的培养基上生长,表明该菌中存在琥珀酰亚胺完整的转化途径。从112R4基因组DNA出发,用鸟枪法克隆了一个6kb的与环酰亚胺水解相关的DNA片段;进一步亚克隆得到了带酰亚胺酶基因的2kb的DNA片段,并进行了序列测定。缺失分析确定了一个876bp的ORF为真养产碱杆菌112R4的酰亚胺酶基因,推测编码一个291个氨基酸的多肽,这是第一次报道微生物酰亚胺酶的核酸和蛋白序列。推测的氨基酸序列在蛋白数据库中进行了比较,结果表明,酰亚胺酶与已知的环酰胺酶没有明显的同源性,也不属于氨酰水解酶蛋白超家族,因而被分类为一种新的环酰胺酶。真养产碱杆菌112R4的酰亚胺酶与芽生杆菌A17p4的酰亚胺酶N端的20个氨基酸有较高的同源性,一致性为60%,与多糖脱乙酰酶保守序列也部分同源,一致性为14%。带有酰亚胺酶基因的重组质粒在大肠杆菌中得到表达,在lac启动子控制下,使用1mmol/L IPTG诱导5h,酰亚胺酶活力达到3200U/L,为供体菌真养产碱杆菌112R４的7倍。     

Engineering a disulfide bond and free thiols in the lantibiotic nisin Z.

The antimicrobial peptide nisin contains the uncommon amino acid residues lanthionine and methyl-lanthionine, which are post-translationally formed from Ser, Thr and Cys residues. To investigate the importance of these uncommon residues for nisin activity, a mutant was designed in which Thr13 was replaced by a Cys residue, which prevents the formation of the thioether bond of ring C. Instead, Cys13 couples with Cys19 via an intramolecular disulfide bridge, a bond that is very unusual in lantibiotics. NMR analysis of this mutant showed a structure very similar to that of wild-type nisin, except for the configuration of ring C. The modification was accompanied by a dramatic reduction in antimicrobial activity to less than 1% of wild-type activity, indicating that the lanthionine of ring C is very important for this activity. The nisin Z mutants S5C and M17C were also isolated and characterized; they are the first lantibiotics known that contain an additional Cys residue that is not involved in bridge formation but is present as a free thiol. Secretion of these peptides by the lactococcal producer cells, as well as their antimicrobial activity, was found to be strongly dependent on a reducing environment. Their ability to permeabilize lipid vesicles was not thiol-dependent. Labeling of M17C nisin Z with iodoacetamide abolished the thiol-dependence of the peptide. These results show that the presence of a reactive Cys residue in nisin has a strong effect on the antimicrobial properties of the peptide, which is probably the result of interaction of these residues with thiol groups on the outside of bacterial cells.     

Aqueous Synthesis of Peptide Thioesters from Amino Acids and a Thiol Using 1,1′-Carbonyldiimidazole

A new method was developed for the synthesis of peptide thioesters from free amino acids and thiols in water. This one-pot simple method involves two steps: (1) activation in water of an amino acid presumably as its N-carboxyanhydride (NCA) using 1,1′-carbonyldiimidazole (CDI), and (2) subsequent condensation of the activated amino acid-NCA in the presence of a thiol. With this method citrulline peptide thioesters containing up to 10 amino acid residues were prepared in a single reaction. This aqueous synthetic method provides a simple way to prepare peptide thioesters for studies of peptide replication involving ligation of peptide thioesters on peptide templates. The relevance of peptide replication to the origin-of-life process is supported by previous studies showing that amino acid thioesters (peptide thioester precursors) can be synthesized under prebiotic conditions by reaction of small sugars with ammonia and a thiol.     

Assay and properties of diaminopimelate epimerase from Bacillus megaterium

1. Diaminopimelate epimerase from a soluble extract of Bacillus megaterium N.C.I.B. 7581 was purified about 25-fold by fractionation with ammonium sulphate and chromatography on calcium phosphate gel-cellulose. The product was impure but was unstable on further purification. 2. Quantitative assay methods for the enzyme were devised in which meso- or ll-diaminopimelic acid may be the substrate. 3. Between 25 degrees and 45 degrees at pH7.0 enzyme action leads to an equilibrium mixture containing 65% meso-isomer and 35% ll-isomer. 4. The initial rate of epimerization was 2-3 times as fast with ll-diaminopimelic acid as substrate as with the meso-isomer; a number of other amino acids were not racemized by the enzyme. The Michaelis constants at 37 degrees were 6.7mm (ll-isomer) and 100mm (meso-isomer); with both substrates enzyme activity was maximal at pH7-8. The relative rates of epimerization of ll-diaminopimelic acid at 25 degrees , 37 degrees and 45 degrees were 0.77:1.00:1.15. 5. A thiol compound (of which 2,3-dimercaptopropan-1-ol was the most effective) was needed as an activator of the purified enzyme. 6. Carbonylbinding reagents and several other compounds did not inhibit diaminopimelate epimerase. 7. Pyridoxal phosphate did not stimulate enzymic activity even in preparations that had been almost completely freed of derivatives of vitamin B(6) (as shown by microbiological assay).     

Isolation and characterization of porcine somatotropin containing a succinimide residue in place of aspartate129.

Aspartate129 in porcine somatotropin was converted into a cyclic imide residue (succinimide) under acidic solution conditions. Reversed-phase high performance liquid chromatography was utilized to isolate and quantitate this altered species, which accounted for approximately 30% of the total protein. The molecular mass of this modified species was determined by electrospray mass spectrometry to be 18 Da less than normal porcine somatotropin, indicative of a loss of 1 H2O molecule. Tryptic peptide mapping demonstrated that the peptide composed of residues 126-133 was altered in this modified protein. Amino acid analysis, amino acid sequencing, mass spectrometry, and capillary zone electrophoresis were used to demonstrate that aspartate129 in this peptide had been converted into a succinimide residue. Further confirmation that this peptide contained a succinimide was obtained by hydrolyzing the modified peptide at pH 9.0, which yielded both the aspartate and isoaspartate peptides.     

The subunit structure of rabbit skeletal-muscle phosphofructokinase and the amino acid sequence of the tryptic peptide containing the highly reactive thiol group.

1. The single highly reactive (class I) thiol group per 80000-mol.wt. subunit of skeletal-muscle phosphofructokinase was specifically carboxymethylated with iodo[2-14C]acetate, and after denaturation the remaining thiol groups were carboxymethylated with bromo[2-3H]acetate. After tryptic digestion and peptide 'mapping' it was found that the 14C radioactivity was in a spot that did not contain significant amounts of 3H radioactivity, so it is concluded that there is not a second, 'buried' cysteine residue within a sequence identical with that of the class-I cysteine peptide. 2. The total number of tryptic peptides as well as the number of those containing cysteine, histidine or tryptophan were inconsistent with the smallest polypeptide chain of phosphofructokinase (mol.wt. about 80000) being composed of two identical amino acid sequences. 3. The amino acid sequence of the tryptic peptide containing the class-I thiol group was shown to be Cys-Lys-Asp-Phe-Arg. This sequence is compared with part of the sequence containing the highly reactive thiol group of phosphorylase.     

Post-translational amino acid isomerization: a functionally important D-amino acid in an excitatory peptide

The post-translational modification of an L- to a D-amino acid has been documented in relatively few gene products, mostly in small peptides under 10 amino acids in length. In this report, we demonstrate that a 46-amino acid polypeptide toxin has one D-phenylalanine at position 44, and that the epimerization from an L-Phe to a D-Phe has a dramatic effect on the excitatory effects of the peptide. In one electrophysiological assay carried out, the D-Phe-containing peptide was extremely potent, whereas the unmodified polypeptide had no biological activity, demonstrating that the chirality of the post-translationally modified amino acid is functionally significant. The peptide toxin analyzed, r11a, belongs to the I-gene superfamily of conotoxins that has four disulfide cross-links. The D-Phe in r11a is at the third amino acid from the C terminus, the same relative position from the C-terminal end as the d-amino acid in omega-agatoxin TK from a spider, an unrelated peptide. Thus, although post-translational amino acid isomerization appears to have no strong specificity for the chemical nature of the amino acid side chain, the few peptides where this modification has been established suggest that there may be favored positions near the N or C terminus that are preferential sites for isomerization to a D-amino acid.     

An important side reaction using the thiol, 3,6‐dioxa‐1,8‐octanedithiol (DODT), in 9‐fluorenylmethoxycarbonyl‐based solid phase peptide synthesis

A considerable quantity of an alkylation by‐product is observed when using 3,6‐dioxa‐1,8‐octanedithiol as a scavenger during acidic release of peptides containing the thioether amino acid methionine from the solid support. Adjustment of the cleavage conditions by replacement of 3,6‐dioxa‐1,8‐octanedithiol with ethane dithiol or by using methionine sulfoxide as an alternative to methionine resulted in no such impurity. The by‐product was detectable by liquid chromatography and mass spectrometry and characterised by NMR spectroscopy of an isolated model peptide. It could be effectively removed in a separate post cleavage step by treatment with dilute aqueous acid at 37 °C. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Adrenomedullin disulfide bond mimetics uncover structural requirements for AM1 receptor activation

Adrenomedullin (ADM) is a vasoactive peptide hormone of 52 amino acids and belongs to the calcitonin peptide superfamily. Its vasodilative effects are mediated by the interaction with the calcitonin receptor‐like receptor (CLR), a class B G protein‐coupled receptor (GPCR), associated with the receptor activity modifying protein 2 (RAMP2) and functionally described as AM‐1 receptor (AM₁R). A disulfide‐bonded ring structure consisting of six amino acids between Cys¹⁶ and Cys²¹ has been shown to be a key motif for receptor activation. However, the specific structural requirements remain to be elucidated. To investigate the influence of ring size and position of additional functional groups that replace the native disulfide bond, we generated ADM analogs containing thioether, thioacetal, alkane, and lactam bonds between amino acids 16 and 21 by Fmoc/t‐Bu solid phase peptide synthesis. Activity studies of the ADM disulfide bond mimetics (DSBM) revealed a strong impact of structural parameters. Interestingly, an increased ring size was tolerated but the activity of lactam‐based mimetics depended on its position within the bridging structure. Furthermore, we found the thioacetal as well as the thioether‐based mimetics to be well accepted with full AM₁R activity. While a reduced selectivity over the calcitonin gene‐related peptide receptor (CGRPR) was observed for the thioethers, the thioacetal was able to retain a wild–type‐like selectivity profile. The carbon analog in contrast displayed weak antagonistic properties. These results provide insight into the structural requirements for AM₁R activation as well as new possibilities for the development of metabolically stabilized analogs for therapeutic applications of ADM.     

Characterization of cysteine thiol modifications based on protein microenvironments and local secondary structures

We have demonstrated earlier that protein microenvironments were conserved around disulfide‐bridged cystine motifs with similar functions, irrespective of diversity in protein sequences. Here, cysteine thiol modifications were characterized based on protein microenvironments, secondary structures and specific protein functions. Protein microenvironment around an amino acid was defined as the summation of hydrophobic contributions from the surrounding protein fragments and the solvent molecules present within its first contact shell. Cysteine functions (modifications) were grouped into enzymatic and non‐enzymatic classes. Modifications studied were—disulfide formation, thio‐ether formation, metal‐binding, nitrosylation, acylation, selenylation, glutathionylation, sulfenylation, and ribosylation. 1079 enzymatic proteins were reported from high‐resolution crystal structures. Protein microenvironments around cysteine thiol, derived from above crystal structures, were clustered into 3 groups—buried‐hydrophobic, intermediate and exposed‐hydrophilic clusters. Characterization of cysteine functions were statistically meaningful for 4 modifications (disulfide formation, thioether formation, sulfenylation, and iron/zinc binding) those have sufficient amount of data in the current dataset. Results showed that protein microenvironment, secondary structure and protein functions were conserved for enzymatic cysteine functions, in contrast to the same function from non‐enzymatic cysteines. Disulfide forming enzymatic cysteines were tightly packed within intermediate protein microenvironment cluster, have alpha‐helical conformation and mostly belonged to CxxC motif of electron transport proteins. Disulfide forming non‐enzymatic cysteines did not belong to conserved motif and have variable secondary structures. Similarly, enzymatic thioether forming cysteines have conserved microenvironment compared to non‐enzymatic cystienes. Based on the compatibility between protein microenvironment and cysteine modifications, more efficient drug molecules could be designed against cysteine‐related diseases.