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.     

SYNTHESIS OF TWO ANALOGS OF A CYCLIC HEXAPEPTIDE WITH DISULFIDE BRIDGE CORRESPONDING TO BOVINE PROTHROMBIN PRECURSOR SEQUENCE 18–23: Extent of Carboxylation by Vitamin K-Dependent Carboxylase

The synthesis of two analogs of sequence 18–23 of bovine prothrombin precursor is described. Hexapeptides Boc-Cys(Acm)-Leu-Glu(OBzl)-Glu (OBzl)-Pro-Cys(Acm)-OBzl and Ac-Cys(Acm)-Leu-Glu(OBzl)-Glu(OBzl)-Pro-Cys(Acm)-OMe were synthesized in solution by stepwise addition of Boc-amino acids using dicyclohexylcarbodiimide/N-hydroxybenzotriazole as the coupling reagent. The acetamidomethyl groups were cleaved and oxidized, using iodine in methanol, to the protected cyclic disulfide in 62–69% yield. The 0-benzyl groups were removed either by treatment with anhydrous hydrogen fluoride or hydrogen bromide in trifluoroacetic acid to give the cyclic hexapeptide disulfides, R₁-Cys-Leu-Glu-Glu-Pro-Cys-OR₂ where R₁, = H or Ac and R₂ = H or CH₃. The cyclic hexapeptides were evaluated as substrates for vitamin K-dependent carboxylase. Both peptides are unusually poor substrates for the carboxylase, and each appears to inhibit carboxylation of Phe-Leu-Glu-Glu-Leu, a good substrate for the enzyme.     

Synthesis of the C-terminal half of thymosin α1 by the polymeric reagent method

In this report we further show the utility and efficiency of polymer-bound 1-hydroxybenzotriazole (PHBT) as an almost ideal support for the polymeric reagent method of peptide synthesis. This was demonstrated by the synthesis of thymosin α₁ (15–28), in which two suitably blocked segments, Boc-Asp (OtBu)-Leu-Lys (2Cz)-Glu (OBzl)-Lys (2Cz)-Lys (2Cz)-OH ( 3 ) and Boc-Glu (OBzl)-Val-Val-Glu (OBzl)-Glu (OBzl)-Ala-Glu (OBzl)-Asn-OBzl ( 2 ), were prepared entirely by utilizing PHBT activation for each coupling step. After appropriate deblocking of 2 , segments 2 and 3 were coupled by the DCC-HOBT method, followed by complete deblocking and ion-exchange chromatographic purification, affording the C-terminal half of thymosin α₁, H-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH ( 1 ).     

SYNTHESIS BY CONVENTIONAL METHODS OF HUMAN GROWTH HORMONE PEPTIDE FRAGMENTS. I.

The synthesis of two protected peptides which correspond to positions 139–146 and 147–156 of the HGH primary structure is described. These peptides prepared by the stepwise procedure, are: Boc-Phe-Lys(Z)-Gln-Thr(Bzl)-Tyr(Bzl)-Ser(Bzl)-Lys(Z)-Phe-OMe and Boc-Asp(OBzl)-Thr(Bzl)-Asn-Ser(Bzl)-His(Dnp)-Asn-Asp(OBzl)-Asp(OBzl)-Ala-Leu-OBzl. All protected intermediates were isolated and characterized for homogeneity.     

Influence of oligopeptide transporter binding affinity upon uptake and transport of d-Asp(OBzl)-Ala and Asp(OBzl)-Sar in filter-grown Caco-2 monolayers

The oligopeptide transporter, which is responsible for the absorption of various di/tripeptides and several peptidomimetic drugs across the intestinal epithelia, is expressed in mature Caco-2 monolayers. Using certain enzymatically stable dipeptides containing either l- or d-aspartic acid at the amino terminus, we investigated the relationship between a side-chain modified dipeptide's degree of binding affinity for the apically expressed Caco-2 oligopeptide transporter and its ability to undergo uptake and/or apical-to-basal transport. Two β-esterified dipeptides, d-Asp(OBzl)-Ala and Asp(OBzl)-Sar, possess markedly different affinities for the Caco-2 oligopeptide transporter (IC₅₀=2.62±0.35 and 0.014±0.007 mM, respectively) as determined using a [¹⁴C]Gly-Sar cellular uptake displacement assay. d-Asp(OBzl)-Ala undergoes rapid internalization into Caco-2 monolayers (14.33±1.00 nmol/mg protein) during a 15-min uptake study; additionally, d-Asp(OBzl)-Ala is efficiently transported in the apical-to-basal direction across Caco-2 monolayers (14.41±0.91 nmol/h/cm²). Both uptake and transport of d-Asp(OBzl)-Ala are >90% inhibitable by the presence of a 20-fold molar excess of Gly-Pro in the apical chamber. Although Asp(OBzl)-Sar demonstrates a 187-fold lower IC₅₀ value than d-Asp(OBzl)-Ala, Asp(OBzl)-Sar does not achieve uptake or transport in parallel experiments. These data indicate that a side-chain modified, enzymatically stable dipeptide, d-Asp(OBzl)-Ala, is actively taken up into and transported across Caco-2 monolayers via the oligopeptide transporter. Additionally, the degree of affinity of a side-chain modified dipeptide for the Caco-2 oligopeptide transporter is not necessarily indicative of its ability to access the oligopeptide transporter-mediated uptake and transport pathway.     

Synthesis of oligophosphoseryl sequences occurring in casein

The protected oligophosphoseryl peptides from bovine caseins, Z-Xxx-{Ser[PO(OPh)₂]}₃-Glu(OBzl)-OBzl for Xxx = Ile, Val, Gly, Leu and Ph = phenyl, were synthesized in high yields by stepwise lengthening using Boc-Ser[PO(OPh)₂]-OH as acylating carboxyl component and N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride as coupling reagent. The hydrogenolytic deprotection (PtO₂) was carried out with the valine derivative and with the tetrapeptide Ser[PO(OPh)₂]₃-Glu(OBzl)-OBzl. Phosphorylation of oligoseryl peptides failed to give the expected products. Large scale phosphorylation of protected serine was carried out in the presence of triethylamine using absolute ether as a solvent. 2,2,2-Trichloroethyl group (Tc) was shown to be a useful phosphorus protecting moiety in phosphopeptide synthesis: Boc-Ser[PO(OTc)₂]-OBzl, Z-Ser[PO(OTc)₂]-OBzl and Boc-Glu(OBzl)-Ser[PO(OTc)₂]-OBzl were synthesized in high yields using bis-(2,2,2-trichloroethyl) phosphochloridate.     

β-Carboxylic acid esterified d-Asp-Ala retains a high affinity for the oligopeptide transporter in Caco-2 monolayers

d-Asp-Ala, a metabolically stable dipeptide, possesses a relatively high affinity for the Caco-2 oligopeptide transporter (IC₅₀ = 5.75 ± 0.09 mM) as demonstrated by its ability to compete with [¹⁴c]Gly-Sar in cellular uptake experiments. When the β-carboxylic acid of d-Asp-Ala is modified by esterification with a cyclohexyl group (d-Asp(OcHx)-Ala) or a benzyl group (d-Asp(OBzl)-Ala), the resulting compounds are still able to inhibit [¹⁴c]GlySar binding to the oligopeptide transporter, i.e., IC_so values for d-Asp(OcHx)-Ala and d-Asp(OBzl)-Ala were 2.80 ± 0.11 and 2.62 ±0.35 mM, respectively. HPLC analysis shows that both d-Asp-(OcHx)-Ala and d-Asp(OBzl)-Ala are fully resistant to degradation for up to 5 h when incubated in the apical media of confluent Caco-2 monolayers. These results demonstrate that it is possible to covalently modify the side chain of one amino acid in an enzymatically stabilized dipeptide with small, aromatic molecules while enabling them to retain their affinity for the oligopeptide transporter.     

Stability, metabolism and transport of D-Asp(OBzl)-Ala--a model prodrug with affinity for the oligopeptide transporter.

The model prodrug D-Asp(OBzl)-Ala has previously been shown to have affinity and to be transported by the oligopeptide transporter PepT1 expressed in Caco-2 cells. The main objective of the present study was to investigate the aqueous stability of D-Asp(OBzl)-Ala and its in vitro metabolism in different gastrointestinal media arising from rats and humans, as well as in human plasma. The second major aim of the study was to evaluate our previous study in Caco-2 cell culture, by determining the effective intestinal permeability (Peff) of D-Asp(OBzl)-Ala in situ using the single-pass rat perfusion model. The aqueous stability studies show water, general buffer, as well as specific acid and base catalysis of D-Asp(OBzl)-Ala. The degradation of the model prodrug was independent of ionic strength. The half-lives in rat jejunal fluid and homogenate were >3 h. In human gastric and intestinal fluids, the half-lives were >3 h and 2.3+/-0. 03 h, respectively. Using the rat single-pass perfusion technique, the effective jejunal permeability (Peff) of D-Asp(OBzl)-Ala was determined to be high (1.29+/-0.5.10-4 cm/s). The 32 times higher Peff value found in the perfusion model compared to Caco-2 cells is most likely due to a higher functional expression of the oligopeptide transporter. Rat jejuna Peff was reduced by approximately 50% in the presence of well known oligopeptide transporter substrates, such as Gly-Sar and cephalexin. It may be that D-Asp(OBzl)-Ala is primarily absorbed intact by the rat jejunal oligopeptide transporter, since the stability in the intestinal homogenate and fluids was rather high (t1/2>2.3 h).     

Stability and in vitro metabolism of dipeptide model prodrugs with affinity for the oligopeptide transporter.

One approach to increase drug stability and to facilitate oral absorption of low bioavailability drugs may be to design oligopeptide ester prodrugs which are stable in the gastrointestinal tract, are transported via the oligopeptide transporter, and finally release the parent drug molecule into the blood circulation and/or by its site of action. In these kinds of prodrugs the ester linkage may be broken by pH dependent and/or enzyme catalyzed hydrolysis. The objective of the present study was to investigate the degradation mechanism and rate of the model compounds Glu(OBzl)-Sar, D-Glu(OBzl)-Ala and Asp(OBzl)-Sar in aqueous solution and in relevant biological media and to compare these results with those of our previous study of D-Asp(OBzl)-Ala. Furthermore, the resulting aqueous stability and in vitro metabolism data are related to our previous affinity data to evaluate if Glu-Sar, D-Glu-Ala, and Asp-Sar have potential as pro-moieties in these kinds of prodrugs. The degradation rates follow first-order kinetics, show maximun stability at pH 4-5 with maximum half-lives for Asp(OBzl)-Sar, Glu(OBzl)-Sar, and D-Glu(OBzl)-Ala of 115 h, 30 days and 152 days, respectively. The stability was dependent on buffer concentration, temperature, pH, and ionic strength. In biological media such as 80% human plasma, human gastric juice and intestinal fluid, and 10% rat jejunal homogenate at 37 degrees C, the half-lives were greater than 1 h except for the hydrolysis of Glu(OBzl)-Sar in 10% rat jejunal homogenate, where the half-life was approximately 16 min. All the stabilized dipeptides may have potential as drug carriers targeting hPepT1.     

Synthesis and biological activity of Substance P C-terminal hexapeptide and heptapeptide analogues

The analogues [Glu(OBzl)¹¹]SP_6–11 and [Glu(OBzl)¹¹]SP_5–11 of the C-terminal hexapeptide and heptapeptide of Substance P have been synthesized by conventional solution methods. In each analogue the SCH₃ group of Met¹¹ is replaced by the COOCH₂C₆H₅ group. The in vitro activity of both analogues has been determined on three biological preparations: guinea pig ileum (GPI), rat vas deferens (RVD), and rat portal vein (RPV). The selectivity for the different receptors has been studied by utilizing atropine-treated guinea pig ileum (GPI + At). The results showed that both analogues are mainly active on GPI through the NK-1 receptor and that both analogues are equipotent to Substance P.     

Synthesis and solubility properties of peptide fragments of human hemoglobin α-chain (123-136)

The solubility prediction method for protected peptides was successfully applied to relatively small peptide fragments of human hemoglobin α-chain (123-136) which contained various polar amino acid residues such as Asp(OBzl), Glu(OBzl), Lys(Z), Ser(Bzl), and Thr(Bzl). As reported previously for hydrophobic peptides and human proinsulin C-peptide fragments, solubility data indicated that the insolubility of protected peptides having a <P_C > value below 0.90 appeared to begin at the octa- or nonapeptide sequence level and that β-sheet structure played an important role in the insolubility of peptides. When a peptide has a β-sheet structure in the solid state, we can clearly determine the critical chain length for peptide insolubility, the solubility dependence on solvent properties, and the solubility independence of amino acid compositions of peptides.     

Synthesis and interaction with metal ions of cyclic oligopeptides bearing carboxyl groups

Cyclic di- and tetrapeptides bearing carboxyl or carboxylate groups, cyclo[Glu(OBzl)-Glu(OMe)], cyclo [Glu-Glu(OMe)], cyclo(Glu-Glu), cyclo[Glu(OMe)-Pro)₂ and cyclo(Glu-Pro)₂ were synthesized and investigated on the intramolecular interaction of carboxyl side chains in the complexation with metal ions in relation with the conformation. The three kinds of cyclic dipeptides were found to take a flagpole boat conformation. Folded conformation of side chains was predominant for cyclo[Glu(OBzl)-Glu(OMe)] and cyclo[Glu-Glu(OMe)]. However, cyclo(Glu-Glu) took an unfolded conformation. Intramolecular interaction of carboxyl groups was observed neither in free state nor in complexation with metal ions. The intramolecular interaction of carboxyl groups was observed in the case of cyclo(Glu-Pro)₂ in the absence of metal ions added. Cyclo[Glu(OMe)-Pro], and cyclo(Glu-Pro)₂ formed a complex with Ca²⁺ and Ba²⁺ without participation of side chains.     

Synthesis of potent agonists of Substance P by replacement of Met11 with Glu(OBzl) and N-terminal glutamine with Glp of the C-terminal hexapeptide and heptapeptide of Substance P

The analogues [Glp⁶,Glu(OBzl)¹¹]SP_(6-11) and [Glp⁵,Glu(OBzl)¹¹]SP_(5-11)) of the C-terminal hexapeptide and heptapeptide of Substance P have been synthesized by conventional solution methods. In each analogue the N-terminal glutamine has been replaced by pyroglutamic acid, while the COOCH₂C₆H₅ ester group has replaced the SCH₃ group of the Met¹¹ side chain. The in vitro activity of both analogues has been determined on three biological preparations: guinea pig ileum (GPI), rat vas deferens (RVD) and rat portal vein (RPV). The results showed that both analogues are highly potent and selective agonists on GPI through the NK-1 receptor. They are more potent than SP itself, with 1.54 and 1.25 respective values of relative potency on GPI. Their selectivity has been studied by utilizing atropine-treated guinea pig ileum (GPI + At). The analogues showed low activity on RVD and RPV tissues, which represent NK-2 and NK-3 monoreceptor assay, respectively. © Munksgaard 1995.     

SYNTHESIS OF Nα-FORMYL-MET-LEU-PHE-OH: AN INDUCER OF CHEMOTAXIS IN PERITONEAL POLYMORPHONUCLEAR NEUTROPHILS

A chemotactic peptide CHO.Met.Leu.Phe.OH has been synthesized classically using the mixed anhydride procedure. The formyl group was introduced by coupling formic acid in the presence of dicyclohexylcarbodiimide to the partially protected tripeptide. The final product was obtained by treatment of the intermediate CHO.Met.Leu.Phe.OBzl with hydrogen fluoride. The ED₅₀ of the peptide in the Boyden chamber assay was 7 × 10^-11 M; in the lysozyme release assay 2.4 × 10^-10 M and in the β-glucuronidase release assay 2.6 × 10^-10 M. In a radioreceptor assay the ID₅₀ of the peptide was 3.3 × 10^-10 M.     

Side reactions in solid-phase peptide synthesis and their applications

Side reactions in peptide synthesis indicate steps needing improvement as well as opportunities for structural diversification in combinatorial design. Among the side reactions observed in this study, transesterification of Boc-Glu(OBzl) occurred in TMAH-catalyzed resin attachment, leading to Boc-DKKREE(OMe) in solid-phase synthesis of Boc-DKKREE. Acetylation of Boc-Arg(NO₂)-resin occurred during resin capping with Ac₂O/Et₃N, leading to GPR(Ac) in GPR synthesis. His- and Lys-modification occurred during GHRPLDKKREE cleavage from resin by Pd(OAc)₂-catalyzed hydrogenation in DMF. To verify these side reactions, model experiments were performed, which indicated rapid transesterification of Boc-Glu(OBz1) in methyl. isopropyl, or tert-butyl alcohol into the corresponding ester by TMAH, but not by Cs. This TMAH ability was used to devise a convenient procedure for peptide cleavage. TLC studies of acetylation showed that both Boc-Arg(NO₂) and Boc-Arg(Tos) were stable to Ac-Im treatment, but were modified by Ac₂O/Et₃N. Since transfer hydrogenation of Boc-His(Bzl) and Boc-Lys(Z) in HCOOH or ammonium formate did not generate the formylated side-products of catalytic hydrogenation, DMF and not its decomposed product, HCOOH, appeared involved in side-chain modification. Elimination of the side reactions, by using Cs-derived Boc-Glu(OBzl)-resin for peptide synthesis and catalytic hydrogenation in NMP-HOPr for peptide cleavage. increased the GHRPLDKKREE yield by 1/3. On the other hand, the side reactions provided modified peptides, whose bioassays revealed different importance of the modified side-chains. © Munksgaard 1996.     

Synthesis of O-glycosylated tuftsins by utilizing threonine derivatives containing an unprotected monosaccharide moiety

Synthesis is described of four tuftsin derivatives containing a D-ghcopyranosyl or a D-galactopyranosyl unit covalently linked to the hydroxy side chain function of the threonine residue through either an α or βO-glycosidic linkage. Fmoc-threonine derivatives containing the suitable unprotected sugar were used for incorporating the O-glycosylated amino acid residue. Z-Thr[α-Glc(OBzl)₄]-OBzl and Z-Thr[α-Gal(OBzl)₄]-OBzl were prepared from the tetra-O-benzylated sugar and Z-Thr-OBzl by the trichloroacetimidate method in the presence of trimethylsilyl trifluoromethane sulfonate. The α glycosylated threonine derivatives were converted into Fmoc-Thr(α-G1c)-OH and Fmoc-Thr(α-Gal)-OH by catalytic hydrogenation followed by acylation with Fmoc-OSu. β-Glucosylation and β-galactosylation of threonine were carried out by reacting the proper per-O-acetylated sugar with Z-Thr-OBzl and boron trifluoride ethyl etherate in dichloromethane. Catalytic hydrogenation of the β-O-glycosylated threonine derivatives followed by acylation with Fmoc-OSu and deacetylation with methanolic hydrazine yielded Fmoc-Thr(β-Glc)-OH and Fmoc-Thr(β-Gal)-OH, respectively. The O-glycosylated threonine derivatives were then reacted with H-Lys(Z)-Pro-Arg(NO₂)-OBzl in the presence of DCC and HOBt and the resulting glycosylated tuftsin derivatives were fully deblocked by catalytic hydrogenation, purified by HPLC, and characterized by optical rotation, amino acid analysis, and ¹H NMR. The β-galactosylated tuftsin was also prepared by the continuous flow solid phase procedure.     

STUDIES ON PEPTIDES LXXXI. Application of a New Arginine Derivative,NG-Mesitylene-2-Sulfonylarginine,to the Synthesis of Substance P and Neurotensin

A new devised arginine derivative, N^G-mesitylene-2-sulfonylarginine, Arg(Mts), was employed for the synthesis of hypothalamic substance P and neurotensin. The former was obtained in 74% yield by treatment of the protected undecapeptide amide, Z - Arg(Mts) - Pro - Lys(Z) - Pro - Gln - Gln - Phe - Phe - Gly - Leu - Met(O)-NH₂, with methanesulfonic acid in the presence of anisole followed by reduction of the sulfoxide with 2-mercaptoethanol. The latter was obtained in 54% yield by the similar treatment of the protected tridecapeptide ester, Z-Pyr-Leu - Tyr - Glu(OBzl) - Asn - Lys(Z)-Pro-Arg(Mts) - Arg(Mts) - Pro - Tyr-Ile-Leu-OBzl, with methanesulfonic acid. As scavenger, a mixture of anisole-thioanisole-o-cresol (1:1:1, by vol.) was employed to suppress the side reaction, O-mesitylene-2-sulfonation of the Tyr residue.     

Synthesis of Met‐enkephalin by solution‐phase peptide synthesis methodology utilizing para‐toluene sulfonic acid as N‐terminal masking of l‐methionine amino acid

The Met‐enkephalin, Tyr‐Gly‐Gly‐Phe‐Met, was synthesized by the solution‐phase synthesis (SPS) methodology employing ‐OBzl group as carboxyls' protection, while the t‐Boc groups were employed for the N‐terminal α‐amines' protection for the majority of the amino acids of the pentapeptide sequence. The l ‐methionine (l ‐Met) amino acid was used as PTSA.Met‐OBzl obtained from the simultaneous protection of the α‐amino, and carboxyl group with para‐toluene sulfonic acid (PTSA) and as‐OBzl ester, respectively in a C‐terminal start of the 2 + 2 + 1 fragments condensation convergent synthetic approach. The protection strategy provided a short, single‐step, simultaneous, orthogonal, nearly quantitative, robust, and stable process to carry through the protected l ‐methionine and l ‐phenylalanine coupling without any structural deformities during coupling and workups. The structurally confirmed final pentapeptide product was feasibly obtained in good yields through the current approach.     

Syntheses of cyclic prodrugs of RGD peptidomimetics with various macrocyclic ring sizes: evaluation of physicochemical,transport and antithrombic properties

Abstract: The objective of this work was to synthesize cyclic prodrugs 1a–d of RGD peptidomimetics 2a–d with various ring sizes (n[CH₂] = 1, 3, 5 and 7) and to evaluate the effect of ring size on their transport, physicochemical, enzymatic stability, and antithrombic properties. The syntheses of cyclic prodrugs 1a–d were achieved by converging two key intermediates, Boc‐Phe‐O‐CH₂‐OCO‐OpNP ( 5 ) and H₂N‐(CH₂)_n‐CO‐Asp(OBzl)‐OTce ( 8a–d ), to give linear precursors Boc‐Phe‐O‐CH₂‐OCO‐HN‐(CH₂)_n‐CO‐Asp(OBzl)‐OTce ( 9a–d ). The N‐ and C‐terminus protecting groups were removed from 9a–d to give 10a–d . Linear precursors 10a–d were cyclized, and the remaining Bzl‐protecting group was removed to produce cyclic prodrugs 1a–d in around 20% overall yield. The linear RGD peptidomimetics ( 2a–d ) were synthesized using standard Boc‐amino acid chemistry by solution‐phase method. Increasing the ring size by adding methylene groups also increases the hydrophobicity of the cyclic prodrugs and parent RGD peptidomimetics. The transport properties of cyclic prodrugs 1c and 1d were 2.6‐ and 4.4‐fold better than those of parent compounds 2c and 2d , respectively. These results suggest that increasing the hydrophobicity of the cyclic prodrugs and parent RGD peptidomimetics enhanced their transport properties. The hydrodynamic radii of the cyclic prodrugs were also smaller than those of their respective parent compounds, suggesting that the change in size may contribute to their transport properties. The chemical stability of the cyclic prodrugs was affected by the ring size, and the cyclic prodrug with the larger ring size (i.e. 1d ) was more stable than the smaller one (i.e. 1a ). All the cyclic prodrugs were more stable at pH 4 than at pH 7 and 10. Prodrug‐to‐drug conversion could be induced by isolated esterase as well as esterase found in human plasma. An increase in the length of methylene group (n[CH₂] = 1, 3, 5, 7) enhanced the antithrombic activity of the prodrugs and the parent compounds. In summary, the ring size of cyclic prodrugs affected their transport, physicochemical, and antithrombic properties.     

Enhancement of peptide coupling reactions by 4-dimethylaminopyridine

4-Dimethylaminopyridine (DMAP) was found to be useful in the enhancement of peptide coupling reactions mediated by dicyclohexylcarbodiimide or symmetrical anhydrides. In an automated synthesis of the model heptapeptide Boc-Ala-Cle-Ile-Val-Pro-Arg(Tos)-Gly-OCH₂-Resin (Cle, cycloleucine), the efficiencies of various coupling methods such as dicyclohexylcarbodiimide, dicyclohexylcarbodiimide plus 1-hydroxybenzotriazole, and symmetrical anhydride were compared with that of dicyclohexylcarbodiimide plus 4-dimethylaminopyridine. Based on the amino acid composition of the peptide-resin samples and high pressure liquid chromatographic analyses of the protected heptapeptide amide obtained from the ammonolytic cleavage of the peptideresin samples, it was concluded that only dicyclohexylcarbodiimide plus 4-dimethylaminopyridine gave the desired near quantitative couplings in those cycles involving the sterically hindered amino acid residues. Observations were also made that 4-dimethylaminopyridine was a useful additive in a modified symmetrical anhydride method of coupling. In the synthesis of the model tetrapeptide Leu-Ala-Gly-Val on a Pam resin, the anhydride couplings were accelerated by DMAP and the product was equivalent in homogeneity to that obtained by the best previous methods. In addition, no racemization was detectable by a sensitive chromatographic method. There also was no detectable racemization found in a DCC-DMAP coupling of Boc-Ile-OH with H-Val-OCH₂-resin. However, significant racemization was observed during the coupling of Boc-Phe-OH with H-Glu(OBzl)-OCH₂-resin. DMAP is recommended as an additive for coupling hindered amino acids, particularly C^α-substituted residues, where little or no racemization is expected.