On the use of N‐dicyclopropylmethyl aspartyl‐glycine synthone for backbone amide protection

To prevent aspartimide formation and related side products in Asp‐Xaa, particularly Asp‐Gly‐containing peptides, usually the 2‐hydroxy‐4‐methoxybenzyl (Hmb) backbone amide protection is applied for peptide synthesis according to the Fmoc‐protocols. In the present study, the usefulness of the recently proposed acid‐labile dicyclopropylmethyl (Dcpm) protectant was analyzed. Despite the significant steric hindrance of this bulky group, N‐terminal H‐(Dcpm)Gly‐peptides are quantitatively acylated by potent acylating agents, and alternatively the dipeptide Fmoc‐Asp(OtBu)‐(Dcpm)Gly‐OH derivative can be used as a building block. In contrast to the Hmb group, Dcpm is inert toward acylations, but is readily removed in the acid deprotection and resin‐cleavage step. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

The aspartimide problem in Fmoc-based SPPS. Part III.

A newly developed Fmoc-Asp derivative, Fmoc-Asp beta-(2,3,4-trimethyl-pent-3-yl) ester, has been tried in the Fmoc-based SPPS of H-Val-Lys-Asp-Xaa-Tyr-Ile-OH, a well-established peptide model for studying base-catalysed aspartimide formation. When synthesizing the hexapeptide incorporating Gly, Arg(Pbf), Asn(Mtt), Asp(OtBu) or Cys(Acm) for Xaa, considerable amounts of aspartimide-related by-products were to be expected. The Asp(3) beta-carboxy protecting group and the duration of exposure to bases were varied. By-product formation could be reduced by incorporation of the new Asp derivative more efficiently than by introducing the less bulky Asp(OMpe). Significant improvements were observed in cases of prolonged contact with piperidine or DBU. Both beta-carboxy protecting groups were superior to the standard Asp(OtBu) which was also included in this study, but the additional stabilization gained by our new protecting group was valuable especially in syntheses of long peptides or difficult sequences.     

The aspartimide problem in Fmoc-based SPPS. Part II.

The sequence dependence of base-catalysed aspartmide formation during Fmoc-based SPPS was systematically studied employing the peptide models H-Val-Lys-Asp-Xaa-Tyr-Ile-OH. The extent of formation of aspartimide and related by-products was determined by RP-HPLC. Considerable amounts of by-products were formed in the case of Xaa = Asp(OtBu), Arg(Pbf), Asn(Mtt), Cys(Acm) and unprotected Thr. Aspartimide formation could be diminished by incorporation of Asp(OMpe) or by employing milder methods for Fmoc cleavage, e.g. hexamethyleneimine/N-methylpyrrolidine/HOBt/NMP/DMSO 4:50:4:71:71 (v/v/w/v/v).     

The aspartimide problem persists: Fluorenylmethyloxycarbonyl‐solid‐phase peptide synthesis (Fmoc‐SPPS) chain termination due to formation of N‐terminal piperazine‐2,5‐diones

Aspartimide (Asi) formation is a notorious side reaction in peptide synthesis that is well characterized and described in literature. In this context, we observed significant amounts of chain termination in Fmoc‐SPPS while synthesizing the N‐terminal Xaa‐Asp‐Yaa motif. This termination was caused by the formation of piperazine‐2,5‐diones. We investigated this side reaction using a linear model peptide and independently synthesizing its piperazine‐2,5‐dione derivative. Nuclear magnetic resonance (NMR) data of the side product present in the crude linear peptide proves that exclusively the six‐membered ring is formed whereas the theoretically conceivable seven‐membered 1,4‐diazepine‐2,5‐dione is not found. We propose a mechanism where nucleophilic attack of the N‐terminal amino function takes place at the α‐carbon of the carbonyl group of the corresponding Asi intermediate. In addition, we systematically investigated the impact of (a) different adjacent amino acid residues, (b) backbone protection, and (c) side chain protection of flanking amino acids. The side reaction is directly related to the Asi intermediate. Hence, hindering or avoiding Asi formation reduces or completely suppresses this side reaction.     

Problem of aspartimide formation in Fmoc-based solid-phase peptide synthesis using Dmab group to protect side chain of aspartic acid.

The sequence-dependent, acid- or base-catalysed aspartimide formation is one of the most serious side reactions in solid-phase synthesis of peptides containing aspartic acid. In the present work, we investigated the susceptibility of 4-(N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]amino)benzyl (Dmab), an aspartic acid beta-carboxy side-chain protecting group, for aspartimide formation. As a model, 15-amino acid-residue galanin fragment analogue containing the Asp-Ala motif was used during Fmoc-based solid-phase synthesis. Our study showed a strong tendency of Dmab-protected peptide to form aspartimide with unusual high efficiency. Furthermore, to investigate the susceptibility of Asp-Ala motif for aspartimide formation during the synthesis using Asp(ODmab), a 5-amino acid-residue galanin fragment LGPDA, different types of resin linkers, variety of Fmoc-deprotection conditions and coupling methods were applied.     

New t‐butyl based aspartate protecting groups preventing aspartimide formation in Fmoc SPPS

Obtaining homogenous aspartyl‐containing peptides via Fmoc/tBu chemistry is often an insurmountable obstacle. A generic solution for this issue utilising an optimised side‐chain protection strategy that minimises aspartimide formation would therefore be most desirable. To this end, we developed the following new derivatives: Fmoc‐Asp(OEpe)‐OH (Epe = 3‐ethyl‐3‐pentyl), Fmoc‐Asp(OPhp)‐OH (Php = 4‐n‐propyl‐4‐heptyl) and Fmoc‐Asp(OBno)‐OH (Bno = 5‐n‐butyl‐5‐nonyl). We have compared their effectiveness against that of Fmoc‐Asp(OtBu)‐OH and Fmoc‐Asp(OMpe)‐OH in the well‐established scorpion toxin II model peptide variants H‐Val‐Lys‐Asp‐Asn/Arg‐Tyr‐Ile‐OH by treatments of the peptidyl resins with the Fmoc removal reagents containing piperidine and DBU at both room and elevated temperatures. The new derivatives proved to be extremely effective in minimising aspartimide by‐products in each application. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Application of Dmb-Dipeptides in the Fmoc SPPS of Difficult and Aspartimide-Prone Sequences

Mutter’s pseudoproline dipeptides and Sheppard’s Hmb derivatives are powerful tools for enhancing synthetic efficiency in Fmoc SPPS. They work by exploiting the natural propensity of N-alkyl amino acids to disrupt the formation of the secondary structures during peptide assembly. Their use results in better and more predictable acylation and deprotection kinetics, enhanced reaction rates, and improved yields of crude products. However, these approaches have certain limitations: pseudoproline dipeptides can only be used for sequences containing serine or threonine, and the coupling of the amino acid following the Hmb residue can be extremely difficult. To alleviate some of these shortcomings, we have prepared a range of Fmoc-Aaa-(Dmb)Gly-OH dipeptides and tested their efficacy in the synthesis of a number of challenging hydrophobic peptides. We also compared the efficiency of N-Dmb against N-Hmb backbone protection in preventing aspartimide formation in the Fmoc SPPS of peptides containing the Asp-Gly sequence.     

1,4-diazepine-2,5-dione ring formation during solid phase synthesis of peptides containing aspartic acid beta-benzyl ester.

The Fmoc-based SPPS of H-Xaa-Asp(OBzl)-Yaa-Gly-NH(2) sequences results in side reactions yielding not only aspartimide peptides and piperidide derivatives, but also 1,4-diazepine-2,5-dione-peptides. Evidence is presented to show that the 1,4-diazepine-2,5-dione derivative is formed from the aspartimide peptide. The rate of this ring transformation depends primarily on the tendency to aspartimide and piperidide formation, which is influenced by the nature of the amino acid following the aspartic acid beta-benzyl ester (Xaa). However the bulkiness of the amino acid side chain preceeding the aspartic acid beta-benzyl ester (Yaa) is also important. Under certain conditions the 1,4-diazepine-2,5-dione peptide derivative may even be formed dominantly, which is a highly undesirable side reaction in peptide synthesis, but which provides a new way for the synthesis of diazepine peptide derivatives with targeted biological or pharmacological activity.     

Base-induced side reactions in Fmoc-solid phase peptide synthesis:Minimization by use of piperazine as Nα-deprotectionreagent

Summary Base-induced aspartimide (cyclic imide) and subsequent base adduct formation in the Fmoc-solid phase synthesis of sensitive sequences are serious side reactions that are difficult to both anticipate and control. The effect of extended treatment of piperazine as N^α-Fmoc deprotection reagent on two sensitive peptide sequences was examined. For comparison, other bases were also investigated, including piperidine, 1-hydroxypiperidine, tetrabutylammonium fluoride, and 1,8-diazabicyclo[5.4.0]undec-7-ene. The results showed that all bases induced varying degrees of both aspartimide and, in some cases, base adduct formation, although piperazine caused the least side reaction. Use ofN-(2-hydroxy-4-methoxybenzyl) peptide backbone amide protection was confirmed to confer complete protection against side reaction. In the absence of such protection, for all bases, the use of 1-hydroxybenzotriazole as additive had some, but not complete, beneficial effect in further reducing side reaction. Best results were obtained with piperazine containing 0.1M 1-hydroxybenzotriazole indicating that this reagent merits serious consideration for N^α-deprotection in the Fmoc-solid phase synthesis of base-sensitive sequences. A further advantage of this reagent is that it causes little racemisation of resin-bound C-terminal cysteine, an occasionally serious base-mediated problem in Fmoc-solid phase assembly. A preliminary account of this work was presented at the 25th European Peptide Symposium, Budapest, Hungary, 1998.     

Study of the alkylation propensity of cations generated by acidolytic cleavage of protecting groups in Boc chemistry.

The alkylation of cysteine residue by different classes of carbonium ions, derived from the cleavage of side chain protective groups in anhydrous HF, was investigated. It was found that side chain protection as beta-2,4-dimethylpent-3-yl ester (Dmp) or 2,4-dimethylpent-3-yloxycarbonyl (Doc) groups resulted in more than seven-fold lower level of alkylated byproducts. This makes Dmp and Doc protection of amino acid side chain during solid phase synthesis particularly valuable in the synthesis of peptides containing cysteine residues or other functional groups prone to alkylation by carbonium ions.     

Orthogonal solid-phase synthesis of N-glycopeptides using backbone protection to eliminate aspartimide formation

Summary The side reaction of aspartimide formation during the activation of aspartic acid-containing peptides was eliminated by using backbone protection. The methodology was applied in the orthogonal solid-phase synthesis of N-glycopeptides.     

Preventing aspartimide formation in Fmoc SPPS of Asp‐Gly containing peptides — practical aspects of new trialkylcarbinol based protecting groups

In our efforts to develop a universal solution to the problem of aspartimide formation in Fmoc SPPS, we investigated the application of our new β‐trialkylmethyl protected aspartic acid building blocks to the synthesis of peptides containing the Asp‐Gly motif. The N^α‐Fmoc aspartic acid β‐tri‐(ethyl/propyl/butyl)methyl esters were used in the synthesis of the classic model peptide scorpion toxin II (VKDGYI), and their effectiveness in minimising aspartimide formation during extended piperidine treatments was evaluated. Furthermore, we compared their efficacy against that of the commonly used approach of adding acids to the Fmoc deprotection solution. Finally, we applied our aspartic acid building blocks to the stepwise Fmoc SPPS of teduglutide, a human GLP‐2 analogue, whose synthesis is made challenging by extensive aspartimide formation. In all experiments, our approach led to almost complete reduction of aspartimide formation with accompanied suppression of aspartic acid epimerisation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Secondary structures without backbone: an analysis of backbone mimicry by polar side chains in protein structures.

Backbone mimicry by the formation of closed-loop C7, C10 and C13 (mimics of gamma-, beta- and alpha-turns) conformations through side chain-main chain hydrogen bonds by polar groups is a frequent observation in protein structures. A data set of 250 non-homologous and high-resolution protein crystal structures was used to analyze these conformations for their characteristic features. Seven out of the nine polar residues (Ser, Thr, Asn, Asp, Gln, Glu and His) have hydrogen bonding groups in their side chains which can participate in such mimicry and as many as 15% of all these polar residues engage in such conformations. The distributions of dihedral angles of these mimics indicate that only certain combinations of the dihedral angles involved aid the formation of these mimics. The observed examples were categorized into various classes based on these combinations, resulting in well defined motifs. Asn and Asp residues show a very high capability to perform such backbone secondary structural mimicry. The most highly mimicked backbone structure is of the C10 conformation by the Asx residues. The mimics formed by His, Ser, Thr and Glx residues are also discussed. The role of such conformations in initiating the formation of regular secondary structures during the course of protein folding seems significant.     

Sequence dependence of aspartimide formation during 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis

Summary We have examined the sequence dependence of aspartimide formation during Fmoc-based solid-phase synthesis of the peptide Val-Lys-Asp-X-Tyr-Ile. The extent of aspartimide formation and subsequent conversion to the - or -piperidide was characterized and quantitated by analytical reversed-phase high-performance liquid chromatography and fast atom bombardment mass spectrometry. Aspartimide formation occurred for X=Arg(Pmc), Asn(Trt), Asp(OtBu), Cys(Acm), Gly, Ser, Thr and Thr(tBu). No single approach was found that could inhibit this side reaction for all sequences. The most effective combinations, in general, for minimization of aspartimide formation were (i) tert-butyl side-chain protection of aspartate, piperidine for removal of the Fmoc group, and either 1-hydroxybenzotriazole or 2,4-dinitrophenol as an additive to the piperidine solution; or (ii) 1-adamantyl side-chain protection of aspartate and 1,8-diazabicyclo[5.4.0]undec-7-ene for removal of the Fmoc group.     

Synthesis and monoamine transporter binding properties of 2beta-[3'-(substituted benzyl)isoxazol-5-yl]- and 2beta-[3'-methyl-4'-(substituted phenyl)isoxazol-5-yl]-3beta-(substituted phenyl)tropanes

A series of 2beta-[3'-(substituted benzyl)isoxazol-5-yl]- and 2beta-[3'-methyl-4'-(substituted phenyl)isoxazol-5-yl]-3beta-(substituted phenyl)tropanes were prepared and evaluated for affinities at dopamine, serotonin, and norepinephrine transporters using competitive radioligand binding assays. The 2beta-[3'-(substituted benzyl)isoxazol-5-yl]-3beta-(substituted phenyl)tropanes (3a-h) showed high binding affinities for the dopamine transporter (DAT). The IC(50) values ranged from 5.9 to 22nM. On the other hand, the 2beta-[3'-methyl-4'-(substituted phenyl)isoxazol-5-yl]-3beta-(substituted phenyl)tropanes (4a-h), with IC(50) values ranging from 65 to 173nM, were approximately 3- to 25-fold less potent than the corresponding 2beta-[3'-(substituted benzyl)isoxazol]tropanes. All tested compounds were selective for the DAT relative to the norepinephrine transporter (NET) and serotonin transporter (5-HTT). 3Beta-(4-Methylphenyl)-2beta-[3'-(4-fluorobenzyl)isoxazol-5-yl]tropane (3b) with IC(50) of 5.9nM at the DAT and K(i)s of 454 and 113nM at the NET and 5-HTT, respectively, was the most potent and DAT-selective analog. Molecular modeling studies suggested that the rigid conformation of the isoxazole side chain in 4a-h might play an important role on their low DAT binding affinities.     

Unexpected aspartimide formation during coupling reactions using Asp(OAl) in solid-phase peptide synthesis

Succinimide ring closure is a well-documented side reaction in the synthesis of certain Asp-containing peptides. This side reaction is typically acid- or base-catalyzed, and its occurrence during coupling reactions has not been previously noted. This unforeseen manifestation of aspartimide formation was detected while exploring a new strategy for side-chain to side-chain lactam formation on a solid support to synthesize cyclo[D-Asp2,Dap5]dynorphin A-(1–11) amide. The availability of allyl protecting groups, which provide an additional level of orthogonality in solid-phase peptide synthesis, was very appealing for use in preparing this conformationally constrained analogue. We found that the allyl ester (OAl) was not sufficient protection from this side reaction in this susceptible D-Asp2-Gly3 sequence. Remarkably, the aspartimide formation appeared to occur during the coupling reaction in the absence of base if excess coupling reagent was present.     

The effect of conformation on the solution stability of linear vs. cyclic RGD peptides.

The objective of this study was to evaluate the relationship between conformational flexibility and solution stability of a linear RGD peptide (Arg-Gly-Asp-Phe-OH; 1) and a cyclic RGD peptide (cyclo-(1, 6)-Ac-Cys-Arg-Gly-Asp-Phe-Pen-NH2; 2); as a function of pH. Previously, it was found that cyclic peptide 2 was 30-fold more stable than linear peptide 1. Therefore, this study was performed to explain the increase in chemical stability based on the preferred conformation of the peptides. Molecular dynamics simulations and energy minimizations were conducted to evaluate the backbone flexibility of both peptides under simulated pH conditions of 3, 7 and 10 in the presence of water. The reactive sites for degradation for both molecules were also followed during the simulations. The backbone of linear peptide 1 exhibited more flexibility than that of cyclic peptide 2, which was reflected in the rotation about the phi and psi dihedral angles. This was further supported by the low r.m.s. deviations of the backbone atoms for peptide 2 compared with those of peptide 1 that were observed among structures sampled during the molecular dynamics simulations. The presence of a salt bridge between the side chain groups of the Arg and Asp residues was also indicated for the cyclic peptide under simulated conditions of neutral pH. The increase in stability of the cyclic peptide 2 compared with the linear peptide 1, especially at neutral pH, is due to decreased structural flexibility imposed by the ring, as well as salt bridge formation between the side chains of the Arg and Asp residues in cyclic peptide 2. This rigidity would prevent the Asp side chain carboxylic acid from orienting itself in the appropriate position for attack on the peptide backbone.     

Crystal structure of activated CheY. Comparison with other activated receiver domains

The crystal structure of BeF(3)(-)-activated CheY, with manganese in the magnesium binding site, was determined at 2.4-A resolution. BeF(3)(-) bonds to Asp(57), the normal site of phosphorylation, forming a hydrogen bond and salt bridge with Thr(87) and Lys(109), respectively. The six coordination sites for manganese are satisfied by a fluorine of BeF(3)(-), the side chain oxygens of Asp(13) and Asp(57), the carbonyl oxygen of Asn(59), and two water molecules. All of the active site interactions seen for BeF(3)(-)-CheY are also observed in P-Spo0A(r). Thus, BeF(3)(-) activates CheY as well as other receiver domains by mimicking both the tetrahedral geometry and electrostatic potential of a phosphoryl group. The aromatic ring of Tyr(106) is found buried within a hydrophobic pocket formed by beta-strand beta4 and helix H4. The tyrosine side chain is stabilized in this conformation by a hydrogen bond between the hydroxyl group and the backbone carbonyl oxygen of Glu(89). This hydrogen bond appears to stabilize the active conformation of the beta4/H4 loop. Comparison of the backbone coordinates for the active and inactive states of CheY reveals that only modest changes occur upon activation, except in the loops, with the largest changes occurring in the beta4/H4 loop. This region is known to be conformationally flexible in inactive CheY and is part of the surface used by activated CheY for binding its target, FliM. The pattern of activation-induced backbone coordinate changes is similar to that seen in FixJ(r). A common feature in the active sites of BeF(3)(-)-CheY, P-Spo0A(r), P-FixJ(r), and phosphono-CheY is a salt bridge between Lys(109) Nzeta and the phosphate or its equivalent, beryllofluoride. This suggests that, in addition to the concerted movements of Thr(87) and Tyr(106) (Thr-Tyr coupling), formation of the Lys(109)-PO(3)(-) salt bridge is directly involved in the activation of receiver domains generally.     

Unexpected aspartimide formation during coupling reactions using Asp(OAl) in solid-phase peptide synthesis

Summary Succinimide ring closure is a well-documented side reaction in the synthesis of certain Asp-containing peptides. This side reaction is typically acid-or base-catalyzed, and its occurrence during coupling reactions has not been previously noted. This unforeseen manifestation of aspartimide formation was detected while exploring a new strategy for side-chain to side-chain lactam formation on a solid support to synthesizecyclo[D-Asp², Dap⁵]dynorphin A-(1-11) amide. The availability of allyl protecting groups, which provide an additional level of orthogonality in solid-phase peptide synthesis, was very appealing for use in preparing this conformationally constrained analogue. We found that the allyl ester (OAl) was not sufficient protection from this side reaction in this susceptible D-Asp²-Gly³ sequence. Remarkably, the aspartimide formation appeared to occur during the coupling reaction in the absence of base if excess coupling reagent was present.