Application of N,N-dimethylformamide dineopentyl acetal for efficient anchoring of Nα-9-fluorenylmethyloxycarbonylamino-acids as p-alkoxybenzyl esters in solid-phase peptide synthesis

The attachment of Fmoc-amino acids onto p-alkoxybenzyl alcohol resins via DCC-DMAP coupling suffers from two different problems: formation of dimers and racemization. The use of N,N-dimethylformamide dineopentyl acetal for the preparation of Fmoc-aminoacyloxybenzyl handles is the basis of a safe and efficient anchoring method that avoids both problems.     

In situ generation of Fmoc-amino acid chlorides using bis-(trichloromethyl)carbonate and its utilization for difficult couplings in solid-phase peptide synthesis

Abstract: This paper reports procedures for the straightforward in situ generation of Fmoc-amino acid chlorides using bis-(trichloromethyl)carbonate (BTC) and their utilization for difficult couplings during solid-phase peptide synthesis. The BTC-mediated coupling of all Fmoc-protected proteinogenic amino acids to a large variety of N-alkylated amino acid-peptidyl-resin was studied. The majority of the couplings proceeded with quantitative conversion and without racemization. The utilization of BTC-mediated coupling for facile solid-phase synthesis of backbone cyclic peptides is presented.     

Synthesis of N-carboxyalkyl and N-aminoalkyl functionalized dipeptide building units for the assembly of backbone cyclic peptides

Abstract: To improve the assembly of backbone cyclic peptides, N-functionalized dipeptide building units were synthesized. The corresponding N-aminoalkyl or N-carboxyalkyl amino acids were formed by alkylation or reductive alkylation of amino acid benzyl or tert-butyl esters. In the case of N-aminoalkyl amino acid derivatives the aldehydes for reductive alkylation were obtained from N,O-dimethyl hydroxamates of N-protected amino acids by reduction with LiAlH₄. N-carboxymethyl amino acids were synthesized by alkylation using bromoacetic acid ester and the N-carboxyethyl amino acids via reductive alkylation using aldehydes derived from formyl Meldrums acid. Removal of the carboxy protecting group leads to free N-alkyl amino acids of very low solubility in organic solvents, allowing efficient purification by extraction of the crude product. These N-alkyl amino acids were converted to their tetramethylsilane-esters by silylation with N,O-bis-(trimethylsilyl)acetamide and could thus be used for the coupling with Fmoc-protected amino acid chlorides or fluorides. To avoid racemization the tert-butyl esters of N-alkyl amino acids were coupled with the Fmoc-amino acid halides in the presence of the weak base collidine. Both theN-aminoalkyl and N-carboxyalkyl functionalized dipeptide building units could be obtained in good yield and purity. For peptide assembly on the solid support, the allyl type protection of the branching moiety turned out to be most suitable. The Fmoc-protected N-functionalized dipeptide units can be used like any amino acid derivative under the standard conditions for Fmoc-solid phase synthesis.     

Synthesis of peptides mediated by KOBt

Coupling of Fmoc-amino acid chlorides can be mediated by the potassium salt of 1-hydroxybenzotriazole (KOBt), the reaction being carried out in an organic medium. The use of a base like NaHC03/Na2C03 or DIEA/NMM/pyridine is not necessary. Coupling is fast and racemization free; the work-up, isolation of the product and scale-up are easy. The pentapeptide sequence of Fmoc-[Leu]enkephalin was thus synthesized in the solution phase on a 5 mmol scale without isolation of any intermediate. Acylation of C-protected N-methylamino acid esters by Fmoc-N-methylamino acid chlorides by this procedure is also feasible, as demonstrated by the synthesis of cyclosporin A fragments 4-7 and 8-11. The peptides obtained in high yields were crystalline solids, unlike earlier reports in which they were obtained mostly as oily or foamy intermediates. They showed spectral properties identical with those of the authentic compounds.     

Racemization during peptide coupling with N,N'-bis(2-oxo-3-oxazolidinyl) phosphinic chloride (BOP-Cl)

The extent of racemization under various circumstances is reported for the coupling of the N-protected model dipeptide Z-Gly-Phe to Val-OMe, mediated by N,N'-bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOC-Cl). BOP-Cl can be used for peptide syntheses either in a one-pot reaction or with preactivation. Maximum yields are obtained with 1.2 equiv. BOP-Cl and 1.2–1.5 equiv. amino-nucleophile and 2.2 equiv. mediatorial base in tetrahydrofuran, but the amount of stereomutation is only tolerable with the use of suppressors (4–6%). BOP-Cl/ HOBt (88% yield, 0% racemization) and BOP-Cl/imidazole (96% yield, 1.6% racemization) are the best choices in apolar solvents and HOBt is to be preferred in DMF (89% yield, 2.4% racemization). Most other additives, HOSu, NP, Pfp, ZnCl₂, DMAP, etc., are not good suppressors.     

Unexpected racemization of proline or hydroxy-proline phenacyl ester during coupling reactions with Boc-amino acids

When L-proline or O-benzyl-trans-4–hydroxy-L-proline phenacyl ester was coupled with Boc-amino acids in dimethylformamide using water-soluble carbodiimide (WSCI) in the presence of anhydrous 1-hydroxybenzotriazole (HOBt) as coupling reagents, extensive racemization was observed at the Cα of the proline or hydroxy-proline residue. The extent of racemization was measured by HPLC after the coupling with Boc-L-Leu-OH in the presence or absence of HOBt. The extent of racemization increased when HOBt was added to the reaction mixture, but greatly decreased when it was not, indicating that HOBt was needed for inducing racemization. Almost no racemization was observed when the coupling reaction was carried out by the mixed anhydride procedure in tetrahydrofuran or by the carbodiimide method in dichloromethane without using HOBt. In the case of coupling reactions with ordinary L-amino acid phenacyl esters, no racemization was observed. Examination of some model systems yielded sufficient evidence to prove that HOBt is an efficient catalyst for racemizing proline or hydroxy-proline phenacyl ester not only in the stage of cyclic intermediate formation but also in the opening of the ring structure. Thus, the racemization reaction was found to be closely related to the formation of the cyclic carbinol-amine derivative.     

Propensity for spontaneous succinimide formation from aspartyl and asparaginyl residues in cellular proteins

One mechanism for the spontaneous degradation of polypeptides is the intramolecular attack of the peptide bond nitrogen on the side chain carbonyl carbon atom of aspartic acid and asparagine residues. This reaction results in the formation of succinimide derivatives and has been shown to be largely responsible for the racemization, isomerization, and deamidation of these residues in several peptides under physiological conditions (Geiger, T. & Clarke, S. J. Biol. Chem. 262, 785–794 (1987)). To determine if similar reactions might occur in proteins, I examined the sequence and conformation about aspartic acid and asparagine residues in a sample of stable, well-characterized proteins. There did not appear to be any large bias against dipeptide sequences that readily form succinimides in small peptides. However, it was found that aspartyl and asparaginyl residues generally exist in native proteins in conformations where the peptide bond nitrogen atom cannot approach the side chain carbonyl carbon to form a succinimide ring. These orientations also represent energy minimum states, and it appears that this factor may account for a low rate of spontaneous damage to proteins by succinimide-linked reactions. The presence of aspartic acid and asparagine residues in other conformations, such as those in partially denatured, conformationally flexible regions, may lead to more rapid succinimide formation and contribute to the degradation of the molecule. The possible role of isoimide intermediates, formed by the attack of the peptide oxygen atom on the side chain carboxyl group, in protein racemization, isomerization, and deamidation is also considered.     

Fast and efficient peptide bond formation using bis‐[α,α‐bis(trifluoromethyl)‐benzyloxy]diphenylsulfur. Part I

Abstract: This study towards the development of sulfurane‐based coupling agents shows that bis‐[α,α‐bis(trifluoromethyl)‐benzyloxy]diphenylsulfur (BTBDS) can facilitate rapid amide bond formation between N^α‐urethane‐protected l ‐amino acids and l ‐phenylalanine ethyl ester in the absence of an external base. The corresponding dipeptide esters were obtained in excellent yields and with no detectable racemization, as judged by analysis of the formed dipeptides by chiral‐phase HPLC. In addition, BTBDS‐mediated condensation of benzoyl‐l ‐phenylalanine with l ‐phenylalanine ethyl ester was also investigated. The results indicate that sulfuranes can be useful for application in racemization‐sensitive systems, such as segment condensation.     

Effect of copper(II) chloride on suppression of racemization in peptide synthesis by the carbodiimide method

Copper(II) chloride was found to be an extremely efficient racemization-suppressing additive in the DCC method as compared with the hitherto known ones, by employing the model coupling Z-Gly-l -Val-OH +h-l -Val-OMe in DMF. Although some other copper salts also had a profound effect, copper(II) chloride was the best from the viewpoint of both racemization suppression and coupling efficiency. The effectiveness of copper(II) chloride was further confirmed by employing the EDC-mediated couplings of Z-Gly-containing dipeptides with amino acid esters or dipeptide esters, and those of Z-l -Ala (or l -Val)-l -Val-OH with amino acid esters or dipeptide esters. In almost all the cases studied, no detectable amount (< 0.1 %) of epimer was observed by the HPLC analysis in the presence of copper(II) chloride. This was also the case even with an extremely stringent coupling system Z-l -Pro-l -Val-OH + H-l -Pro-OMe. With reference to the mechanism of racemization suppression, it was found that copper(II) chloride has a strong ability to suppress the racemization of the 5(4H)-oxazolone, which may be formed from an activated carboxyl component during the coupling.     

Preparation of N-9-fluorenylmethoxycarbonylamino acid chlorides from mixed anhydrides by the action of hydrogen chloride1

N-9-Fluorenylmethoxycarbonyl- (Fmoc) amino-acid chlorides have been prepared by reaction of hydrogen chloride on purified mixed Fmoc-amino acid-monoalkyl carbonic acid anhydrides in dichloromethane. The products partially undergo subsequent conversion to the corresponding esters due to the presence of the liberated alcohol, the extent depending on the nature of the alkyl group. Esterification occurred to 5–20% when the alkyl group was isopropyl. Anhydrides of monoisopropenyl carbonic acid which liberate acetone instead of an alcohol gave products uncontaminated with ester. The three components in a reaction mixture could be determined as the reaction progressed by normal phase high-performance liquid chromatography of aliquots, which had been freed of excess hydrogen chloride, on a μPorasil (underivatized silica) column using tert-butanol-hexane (1.5:98.5) as solvent.     

4-Chloromethylphenoxyacetyl polystyrene and polyamide supports for solid-phase peptide synthesis

Two functionalised supports for the solid-phase synthesis of peptides under mild reaction conditions were prepared: 4-chloromethylphenoxyacetamidomethylcopoly (styrene-1%-divinylbenzene) and 4-chloromethylphenoxyacetyl-norleucylpoly(dimethylacrylamide). They were devised in order to avoid the danger of racemization which exists during base-catalyzed esterification of the first protected amino acid to the 4-alkoxybenzyl alcohol resins formerly employed in combination with N^α-9-fluorenylmethoxycarbonyl and tert.-butyl side-chain protecting groups. Esterification of N^α-protected amino acids to the new resins can be achieved easily and without significant levels of racemization by means of their caesium salts, while cleavage from the supports is possible by treatment with trifluoroacetic acid. The 4-chloromethylphenoxyacetyl polystyrene resin was tested by the synthesis of Leu-enkephalin which was cleaved, at the end of the synthesis, from the solid support in 91% yield by 60% trifluoroacetic acid in methylene chloride, and was shown to be more than 99% pure by ion-exchange chromatography and reverse phase high pressure liquid chromatography.     

Racemization during aminolysis of activated esters of N-alkoxycarbonylamino acids by amino acid anions in partially aqueous solvents and a tactic to minimize it

Racemization during the aminolysis of activated esters of N-alkoxycarbonylamino acids by amino acid anions in aqueous dimethylformamide was examined by determining the epimeric products by high-performance liquid chromatography. Partial racemization occurred for a variety of esters, particularly when sodium hydrogen carbonate was used to generate the anion of d -valine. The racemization results from prolonged contact of unconsumed ester with the alkaline medium. Variation of the stoichiometry of reagents for reactions with N-benzyloxycarbonylphenylalanine (Z-Phe) 4-nitrophenyl ester revealed that racemization could be minimized by using Na₂CO₃ as base and a 50% excess of amino acid anion. An efficient synthesis of optically pure Z-l -Phe-D-Val-OH was achieved with a reaction time of 15 min.     

Racemization mechanism of serine dipeptide active ester derivatives*

The racemization of Z-Gly-Ser(Bzl)-OPcp was studied using the isotope effect to distinguish between enolization and 5(4H)-oxazolone mechanism. Labeled Z-Gly-L(α²H)-Ser(Bzl)-OPcp was racemized with NEt₃ in THF to give k_r^2H = 570; similarly the unlabeled Z-Gly-L-Ser(Bzl)-OPcp yielded a racemization rate constant of k_r^H = 720. The k_r^H/k_r^2H ratio of 1.3 indicates that the serine dipeptide active ester derivatives racemize mainly through 5(4H)-oxazolone and to a lesser extent through the enolization mechanism, provided the 5(4H)-oxazolone racemizes much faster than it couples. 5(4H)-Oxazolone was prepared from Z-Gly-L-Ser(Bzl)-OH with DCC. Its first order racemization rate constant in THF is independent of its concentration, indicating that the racemization is not an intermolecular autoracemization. The unimolecular, that is intramolecular self-catalyzed, racemization is not possible based on molecular models. Therefore, it must be a solvent catalyzed bimolecular reaction. Its racemization was instantaneous with NEt₃ while its coupling with H-Val-OMe was found to be 4 times 10^-2M^-1 s^-1, that is k_r± k_c. The k_c/k_r values, which indicate the extent of racemization during coupling, are compared for benzyloxycarbonylamino and benzyloxycarbonylglycylamino acid active esters; the most vulnerable amino acids are His, Cys and Ser. The k_c/k_r values also indicate that for practical synthetic purposes the pentafluorophenyl esters should be preferred over other active esters to minimize racemization.     

Solid-phase synthesis of bombesin by continuous flow procedure using Fmoc-amino acids*

Bombesin has been synthesized by the continuous flow solid-phase procedure on the derivatized Kieselguhr-supported polydimethylacrylamide resin. Preformed Fmoc-amino acid symmetrical anhydrides (Met, Leu, and Arg) and Fmoc-amino acid active esters were used for amine acylation. The Mtr and the Pmc groups have been alternatively used for masking the side chain function of Arg-3. The progress of the synthesis was monitored by different analytical methods including quantitative solid-phase Edman degradation. Cleavage from the resin and simultaneous formation of the C-terminal amide function were achieved with a methanolic ammonia solution yielding indistinguishable crude peptides which have been purified by HPLC and fully characterized. Preliminary pharmacological experiments indicated that the activity of the synthetic peptides is similar to that previously measured for other synthetic bombesins. For comparison bombesin has also been prepared by solid-phase synthesis on 4-methyl benhydrylamine resin using the Boc chemistry. The results of the two strategies are discussed and compared.     

Urethane-protected N-carboxyanhydrides (UNCAs) as unique reactants for the study of intrinsic racemization tendencies in peptide synthesis

We have established that urethane-protected N-carboxyanhydrides (UNCAs) are uniquely suited for the study of intrinsic racemization tendencies in peptide synthesis. The UNCA allows epimerization only by the direct enolization pathway (proton abstraction from the α-carbon) and does not decompose upon epimerization. A protocol employing the quantitative separation and analysis of enantiomeric N-protected amino acid derivatives by chiral HPLC has been developed to measure the intrinsic rate of racemization of UNCAs under widely varying reaction conditions. The influence of the tertiary amine structure, UNCA side chain structure, and solvent were studied. The same protocol was employed to study the intrinsic rate of racemization of N-protected activated amino acid intermediates generated via ‘onium-type’ activating reagents. We have shown that the trends influencing the intrinsic rate of racemization of UNCAs are maintained under the conditions of in situ activations, and are consistent with the trends found in classical studies in the literature. The results are relevant to peptide synthesis both in solution and on solid phase. The intrinsic rate of racemization for any type of activation with any tertiary amine can be measured by this protocol. © Munksgaard 1997.     

Esterification of 9-Fluorenylmethoxycarbonyl-glycosylated serine and cysteine derivatives with an hydroxymethyl resin

Esterification of glycosylated serine and cysteine derivatives with a 4-alkoxybenzyl alcohol (Wang) resin is described. The classical methods of ester bond formation (symmetrical anhydride, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate [TBTU]. /4-dimethylaminopyridine [DMAP]. with or without 1-hydroxybenzotriazole [HOBT]., pentafluorophenyl [Pfp]. esters) gave high percentages of racemization of the glycosylated serine or cysteine residues. To reduce the d -amino acid content, we found that the best results were obtained with the highly efficient MSNT reagent (2,4,6-mesitylenesulfonyl-3-nitro-1,2,4-triazolide), which gave a high yield of substitution of the resin and the lowest percentage of racemization. A difference in behavior was observed between the two amino acids. The glycosylated cysteine derivative always gave lower racemization than the analogous glycosylated serine.     

Racemization during aminolysis of mixed and symmetrical anhydrides of N-alkoxycarbonylamino acids by amino acid anions in aqueous dimethylformamide

Racemization attending the aminolysis of symmetrical (Sy) and mixed (Mx) anhydrides (An) of N-alkoxycarbonylamino acids by amino acid anions in aqueous dimethylformamide has been examined by reversed phase high-performance liquid chromatography analysis of the N-protected dipeptides produced. Racemization occurred in most cases when the anion was generated using 1 equiv. sodium hydrogen carbonate, in most cases for MxAn reactions when the base was sodium carbonate, and only in a few cases for SyAn reactions when the base was sodium carbonate. Less racemization was associated with MxAn reactions when the chloroformate of a secondary alcohol had been used for their generation. The change in chirality is explained on the basis of the formation and racemization of the 2-alkoxy-5(4H)-oxazolone, which is greater in the presence of the weaker base because of incomplete deprotonation of the amino acid zwitter-ion.     

2-Chlorotrityl chloride resin

The esterification of 2-chlorotrityl chloride resin with Fmoc-amino acids in the presence of DIEA is studied under various conditions. High esterification yields are obtained using 0.6 equiv. Fmoc-amino acid/mmol resin in DCM or DCE, in 25 min, at room temperature. The reaction proceeds without by product formation even in the case of Fmoc-Asn and Fmoc-Gln. The quantitative and easy cleavage of amino acids and peptides from 2-chlorotrityl resin, by using AcOH/TFE/DCM mixtures, is accomplished within 15-60 min at room temperature, while t-butyl type protecting groups remain unaffected. Under these exceptionally mild conditions 2-chlorotrityl cations generated during the cleavage of amino acids and peptides from resin do not attack the nucleophilic side chains of Trp. Met, and Tyr.     

Application of 2-chlorotrityl resin in solid phase synthesis of (Leu15)-gastrin I and unsulfated cholecystokinin octapeptide

The carboxyl terminal dipeptide amide, Fmoc-Asp-Phe-NH₂, of gastrin and cholecystokinin (CCK) has been attached in high yield through its free side chain carboxyl group to the acid labile 2-chlorotrityl resin. The obtained peptide resin ester has been applied in the solid phase synthesis of partially protected (Leu¹⁵)-gastrin I utilising Fmoc-amino acids. Quantitative cleavage of this peptide from resin, with the t-butyl type side chain protection intact is achieved using mixtures of acetic acid/trifluoroethanol/dichloro-methane. Under the same conditions complete detritylation of the tyrosine phenoxy function occurs simultaneously. Thus, the solid-phase synthesis of peptides selectively deprotected at the side chain of tyrosine is rendered possible by the use of 2-chlorotrityl resin and Fmoc-Tyr(Trt)-OH. The efficiency of this approach has been proved by the subsequent high-yield synthesis of three model peptides and the CCK-octapeptide.     

Synthesis of different types of dipeptide building units containing N‐ or C‐terminal arginine for the assembly of backbone cyclic peptides*

Abstract: Different types of dipeptide building units containing N‐ or C‐terminal arginine were prepared for synthesis of the backbone cyclic analogues of the peptide hormone bradykinin (BK: Arg‐Pro‐Pro‐Gly‐Phe‐Ser‐Pro‐Phe‐Arg). For cyclization in the N‐terminal sequence N‐carboxyalkyl and N‐aminoalkyl functionalized dipeptide building units were synthesized. In order to avoid lactam formation during the condensation of the N‐terminal arginine to the N‐alkylated amino acids at position 2, the guanidino function has to be deprotected. The best results were obtained by coupling Z‐Arg(Z)₂‐OH with TFFH/collidine in DCM. Another dipeptide building unit with an acylated reduced peptide bond containing C‐terminal arginine was prepared to synthesize BK‐analogues with backbone cyclization in theC‐terminus. To achieve complete condensation to the resin and to avoid side reactions during activation of the arginine residue, this dipeptide unit was formed on a hydroxycrotonic acid linker. HYCRAM^? technology was applied using the Boc‐Arg(Alloc)₂‐OH derivative and the Fmoc group to protect the aminoalkyl function. The reduced peptide bond was prepared by reductive alkylation of the arginine derivative with the Boc‐protected amino aldehyde, derived from Boc‐Phe‐OH. The best results for condensation of the branching chain to the reduced peptide bond were obtained using mixed anhydrides. Both types of dipeptide building units can be used in solid‐phase synthesis in the same manner as amino acid derivatives.