Preferred conformation of the benzyloxycarbonyl-amino group in peptides*

Structural parameters, derived from X-ray crystallographic data, have been compiled for 35 derivatives of amino acids, peptides, and related compounds, which contain the N-terminal benzyloxycarbonyl (Z) group. The geometry of the urethane moiety of this end group is closely similar to that of the tert-butoxycarbonyl (Boc) group, except for a relaxation of some bond angles because the Z group is sterically less crowded than the Boc group. For the same reason, the Z group has greater conformational flexibility. As a result, packing forces in the crystal may cause greater deformations of bond angles, resulting in larger variations of observed bond lengths and bond angles than in Boc-peptide crystals. The aromatic rings of the Z end groups tend to stack in crystals. Conformational energy calculations indicate that most conformations of Z-amino acid-N' -methylamides and of corresponding Boc derivatives have similar dihedral angles and relative energies, i.e. the nature of the N-terminal end group has little effect on the conformational preferences of the residue next to it. In particular, the computed fraction of molecules with a cis urethane (C-N) bond is similar for the two derivatives: 0.51 and 0.42 in Boc-Pro-NHCH₃ and Z-Pro-NHCH₃, respectively, and 0.02 in the two Ala derivatives. There exist several computed conformations of Z-Ala-NHCH₃ and Z-Pro-NHCH₃ in which the phenyl ring and the C-terminal methylamide group are close to each other. Because of favorable nonbonded interactions, such conformations are of low energy.     

Conformational analysis of cyclic tetrapeptides by molecular mechanics calculations

Molecular mechanics calculations of the cyclic tetrapeptide ring system for the cis, trans, cis, trans amide bond sequences for cyclo tetraglycine and cyclo tetraalanine have been carried out. A systematic search of conformational space was carried out by using Still's RINGMAKER in an attempt to find the global minimum for each amide bond sequence. Ring system structures were optimized by using the BAKMOD program. A comparison of 11 experimentally determined cyclic tetrapeptide conformations with the lowest energy calculated conformations showed that only 4 of 11 known cyclic tetrapeptides adopted the lowest energy conformation. However, when the destabilization energy between cyclo tetraalanine and cyclo tetraglycine was calculated, 10 of the 11 experimentally determined conformations for cyclic tetrapeptides with alternating cis, trans, cis, trans amide bond sequences adopted the conformations with the least de-stabilization energy. The relationship between the molecular mechanics calculations and empirical rules for predicting cyclic tetrapeptide conformations is discussed.     

Use of Fmoc-N-(2-hydroxy-4-methoxybenzyl) amino acids in peptide synthesis

The use of N, O-bisFmoc-N-(2-hydroxy-4-methoxybenzyl) amino acid derivatives in the synthesis of peptides with difficult sequences has already been described. With these amino acid derivatives the reversible protecting group 2-hydroxy-4-methoxybenzyl (Hmb) for the backbone amide bonds of peptide chains is introduced, and thus the aggregation due to hydrogen-bond interchain association is inhibited. This paper describes the synthesis and use of Fmoc-N-(2-hydroxy-4-methoxybenzyl)amino acid derivatives as an alternative means of introducing Hmb backbone protection. These new monoFmoc derivatives were obtained in higher yield than the bisFmoc derivatives. Coupling yields to the amino peptide resin were the same as those obtained with bisFmoc derivatives, under the TBTU/HOBt/DIEA conditions. We also compared different syntheses of a difficult peptide with the Fmoc approach [triple coupling, capping, use of chaotropic agents, backbone protection using monoFmoc (Hmb)Ala] and with optimized Boc chemistry. Both the backbone protection and optimized Boc chemistry approaches gave the desired product in excellent yield and purity. © Munksgaard 1997.     

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.     

5,5-Dimethylthiazolidine-4-carboxylic acid (DTC) as a proline analog with restricted conformation

Spectroscopic evidence is presented for the lack of intramolecular hydrogen bonding in a simple peptide derivative of 5,5-dimethylthiazolidine-4-carboxylic acid (Dtc). The infrared spectrum of Boc-Pro-Ile-OMe 1 in nonpolar solvents displays two N-H stretching bands at 3419 and 3330 cm^-1 in CCl₄ and one at 3417 and 3328cin^-1 in CHCl₃. The low frequency band at 3328–3330cm^-1 may be assigned to conformations with an intramolecular hydrogen bond between the Ile N-H and Boc C=O. The band at 3417-3419 cm^-1 is the normal Ile N-H stretch. In the polar solvent CH₃ CN only one NH stretching band at 3365 cm^-1 is observed. The IR spectrum of Boc-Dtc-Ile-OMe 2, on the other hand, displays one N-H stretching band at 3423cm^-1 in CCI, and one at 3418cm^-1 in CHCI₃. The IR spectrum of 2 does not display the N-H stretching band that would arise from intramolecular hydrogen bonding between the Boc C=O and Ile N-H. The lack of intramolecular hydrogen bonding for Boc-Dtc-Ile-OMe 2 was evident also in the NMR spectra in nonpolar solvents. The ¹H-NMR spectrum of the Pro dipeptide 1 in 50% CDCl₃/C₆D₆ at 20° displayed two Ile-NH signals at 6.58 and 7.74 ppm. The latter signal corresponds to the intramolecularly hydrogen bonded Ile-NH in the trans-Boc isomer of 1 (60% of the total population), while the former signal corresponds to the nonhydrogen bonded Ile-NH in the cis-Boc isomer. The ¹H-NMR spectrum of the Dtc dipeptide 2 displayed two slowly exchanging cis- and trans-Boc amide isomers as well, but both amide proton resonances were observed upfield at 6.67 and 6.74 ppm, which correspond only to a nonhydrogen bonded Ile N-H. The X-ray crystal structure of Boc-Dtc displays only a cis-Boc-Dtc urethane amide group and two conformations for the Dtc ring, one in which the beta carbon atom is anti to the carboxyl group and the other in which the gamma sulfur atom is anti to the carboxyl group. Conformational analysis of Ac-Dtc-NHMe suggests that in the hydrogen bonded C7 conformation steric interaction between the syn-beta methyl group and carbonyl group of Dtc adds nonbonded and angle strain energies to counteract the stabilizing coulombic interaction between the Boc C=O and terminal amide N-H. Whereas the C7 conformation is a prominent conformation for peptide derivatives of proline, other conformations are favored in peptide derivatives of Dtc (ψ -ñ 110-150° or ñ 320-360°). These results suggest that, in peptides where substitution of Pro appears to maintain or enhance biological activity, the substitution of Dtc for Pro may test the functional importance of the C7 conformation in that position of the peptide sequence.     

Chemical mechanism to account for artifactual formation of shortened peptides with free alpha-amino groups in solid phase peptide synthesis

The formation of terminated peptides with free α-amino groups has often been observed in stepwise solid phase peptide synthesis. This has been attributed to variable accessibility in regions of the swollen crosslinked resin supports. It is now shown that impurities in the amino acid reagents are responsible for these by-products. Thus, sec. -butyloxycarbonylamino acids were isolated from tert. -butyloxycarbonylamino acids after treatment with trifluoroacetic acid under standard deprotection conditions for the removal of the tert. -butyloxycarbonyl (Boc) group. Direct reverse phase HPLC analysis of Boc-amino acids from commercial sources also showed the sec. -Boc-amino acids as impurities present at varying levels. The sec. -Boc group was stable to treatment at room temperature with trifluoroacetic acid in dichloromethane (1:1, v/v) (half-life 7 years), but was removed by HF-anisole under the standard conditions of cleavage and deprotection of assembled peptides. In model syntheses, the level of terminated free peptides corresponded to the level of preexisting sec. -Boc-amino acid impurities present in the Boc-amino acid reagents. Use of Boc-amino acids with no detectable sec. -Boc resulted in negligible levels (< 0.05%) of terminated peptides. The problem is thus readily overcome by the use of pure Boc-amino acid starting materials and is not a reflection of a shortcoming inherent to the polymer supported nature of solid phase syntheses as has been previously suggested.     

Conformational study of cyclosporin A in acetone at low temperature

The conformation of cyclosporin A (CsA), an undecapeptide with seven N-methylated amino acids, was studied in acetone at 193 K. Previous studies of the conformation of CsA in different solvents, in the cyclosporin-cyclophilin complex and in complexes with LiCl showed that the conformation of the free and the bound CsA are different. Differences were observed at the conformation of the MeLeu⁹-MeLeu¹⁰ peptide bond, which is cis in solution and trans in the complex, and in the orientation of the amide protons and the N-Me groups. By using acetone, which is a proton acceptor, we wanted to influence the orientation of the amide protons. In the conditions used in this study a new conformation is found, which differs as well from the one previously observed in solution as from the Conformation observed in the complex. This conformation has a cis peptide bond between MeLeu⁹ and MeLeu¹⁰. The trans conformation of the peptide bond MeLeu⁹-MeLeu¹⁰, which is necessary for biological activity, was not induced. One of the amide protons is involved in an intramolecular H-bridge stabilising a β-turn around Sar³MeLeu⁴, and three of the seven NMe groups are oriented to the centre of the molecule. © Munksgaard 1994.     

3-NITRO-2-PYRIDINESULFENYL (Npys) GROUP

The novel 3-nitro-2-pyridinesulfenyl (Npys) group, which is useful for the protection and the activation of amino and hydroxyl groups for peptide synthesis, is reported. The Npys group is readily introduced by treatment of amino acids with 3-nitro-2-pyridinesulfenyl chloride. The Npys group is easily removed by treatment with very dilute HCl, e.g. 0.1-0.2 N HCl in dioxane, but it is resistant to trifluoroacetic acid and 88% formic acid. Npys is also selectively removed under neutral conditions using triphenylphosphine or 2-pyridinethiol 1-oxide without affecting benzyloxycarbonyl (Z), tert-butyloxycarbonyl (Boc), 2-(4-biphenylyl) propyl(2) oxycarbonyl (Bpoc), 9-fluorenylmethyloxycarbonyl (Fmoc), benzyl (Bzl) or tert-butyl (tBu) protecting groups. The N-Npys and O-Npys groups when activated in the presence of RCOOH by the addition of tertiary phosphine form peptide or ester bonds via oxidation-reduction condensation. The important features of the Npys group are demonstrated through the synthesis of peptides in solution and by solid phase methodology without a formal deprotection procedure. In solid phase synthesis, 4-(Npys-oxymethyl) phenylacetic acid is used as the key intermediate for the introduction of the trifluoroacetic acid resistant 4-(oxymethyl) phenylacetamido linking group to the resin.     

CONFORMATIONAL ANALYSIS OF N-(tert.-AMYLOXYCARBONY-l-PROLINE IN THE SOLID STATE AND IN SOLUTION

The solid-state conformational analysis of t-AOC-l -Pro-OH** has indicated that the molecules are not folded up to form an oxy-C₇ peptide conformation, but rather that they are held together through intermolecular O-H. 0 = C (urethane) hydrogen bonds. The tertiary amide bond is in the cis configuration. In solvents of high polarity strongly solvated species largely predominate. In cyclohexane solution non-associated and associated (involving the carboxyl C = O as the proton acceptor) species are simultaneously present. Obviously, the extent of association increases with increasing solute concentration. The amount of the oxy-C₇ form, if any, should be extremely small. It is also demonstrated that CD measurements alone can lead to an incorrect picture of the conformational preferences of amino acid derivatives and small peptides in solution.     

SYNTHESIS OF p-AMINO-L-PHENYLALANINE DERIVATIVES WITH PROTECTED p-AMINO GROUP FOR PREPARATION OF p-AZIDO-L-PHENYLALANINE PEPTIDES

For the synthesis of p-azidophenylalanine peptides, the p-amino group of p-amino-L-phenylalanine is protected with the Z- or Boc residue via the copper complex or by specific acylation at pH 4.6. The α-amino or α-carboxy group is blocked by a protecting group (Boc, Ddz, OMe respectively Z, Nps, Ddz), which can be removed selectively. The synthesis of nine derivatives of p-amino-L-phenylalanine for incorporation into the peptide chain is described. The p-amino-phenylalanine is converted to p-azidophenylalanine without affecting disulfide bridges.     

Structure and solution conformation of the cytostatic cyclic tetrapeptide WF-3161, cyclo[L-leucyl-L-pipecolyl-L-(2-amino-8-oxo-9, 10-epoxydecanoyl)-D-phenylalanyl]

The sequence and configuration of amino acids in the cytostatic cyclic tetrapeptide WF-3161 are established as cyclo(L-Leu-L-Pip-L-Aoe-D-Phe) where Pip = pipecolic acid and Aoe = 2-amino-8-oxo-9,10-epoxydecanoic acid. In chloroform, WF-3161 adopts a conformation with a possible gamma-turn between Leu NH and Aoe C = O and a cis amide bond between Leu and Pip. The torsion angles for this conformation are L-Aoe, phi, -95 degrees, psi, +85 degrees, omega, -155 degrees; D-Phe, phi, +120 degrees, psi, -80 degrees, omega, -175 degrees; L-Leu, phi, -145 degrees, psi, +35 degrees, omega, -10 degrees; L-Pip, phi, +20 degrees, psi, -135 degrees, omega, -170 degrees. The cis,trans,trans,trans amide bond sequence is related to the dimethyl sulfoxide conformation of chlamydocin, another cytostatic cyclic tetrapeptide.     

Synthesis of a tripeptide with a C-terminal nitrile moiety and the inhibition of proteinases

The synthesis of the tripeptide d -Phe-Pro-Arg with the nitrite group instead of the carboxylgroup is described. Initially, the corresponding peptide amide was synthesized by conventional methods in solution using Boc and Fmoc as the protecting group for d -Phe. The dehydration in order to create the nitrite moiety was achieved by treating the peptide amide with phosphorus oxichloride or trifluoroacetic anhydride. Best results were obtained by the use of phosphorus oxichloride in pyridine as the solvent in the presence of imidazole. After deprotection of the N-terminal amino acid the crude product was purified by chromatography on Butyl-Fractogel® HW-40 (S). The purity of the final product was checked on a RP18 phase by hplc. The existence of the nitrite group was demonstrated by i.r. and ¹³C-n.m.r. spectra. The peptide nitrite exhibited a strong inhibition of thrombin compared to the tripeptide amide.     

Proline-containing β-turns

The conformations of the dipeptide t-Boc-Pro-d Ala-OH and the tripeptide tBoc-Pro-d Ala-Ala-OH have been determined in the crystalline state by X-ray diffraction and in solution by CD, n.m.r. and i.r. techniques. The unit cell of the dipeptide crystal contains two independent molecules connected by intermolecular hydrogen bonds. The urethane-proline peptide bond is in the cis orientation in both the molecular forms while the peptide bond between Pro and d Ala is in the trans orientation. The single dipeptide molecule exhibits a “bent” structure which approximates to a partial β-turn. The tripeptide adopts the 4 → 1 hydrogen-bonded type II β-turn with all trans peptide bonds. In solution, the CD and i.r. data on the dipeptide indicate an ordered conformation with an intramolecular hydrogen bond. N.m.r. data indicate a significant proportion of the conformer with a trans orientation at the urethane-proline peptide bond. The temperature coefficient of the amide proton of this conformer in DMSO-d₆ points to a 3 → 1 intramolecular hydrogen bond. Taken together, the data on the dipeptide in solution indicate the presence (in addition to the cis conformer) of a C₇ conformation which is absent in the crystalline state. The spectral data on the tripeptide indicate the presence of the type II β-turn in solution in addition to the nonhydrogen-bonded conformer with the cis peptide bond between the urethane and proline residues. The relevance of these data to studies on the substrate specificity of collagen prolylhydroxylase is pointed out.     

Prodrugs of Peptides. 13. Stabilization of Peptide Amides Against α-Chymotrypsin by the Prodrug Approach

Various derivatives of the C-terminal amide group in N-protected amino acid and peptide amides were synthesized to assess their suitability as prodrug forms with the aim of protecting the amide or peptide bond against cleavage by -chymotrypsin. Whereas N-acetylation, N-hydroxymethylation, and N-phthalidylation did not afford any protection but, in fact, accelerated the terminal amide bond cleavage, condensation with glyoxylic acid to produce peptidyl--hydroxyglycine derivatives and, to a minor extent, N-aminomethylation were found to improve the stability of the parent amides. Besides protecting the terminal, derivatized amide moiety toward cleavage by -chymotrypsin, -hydroxyglycine derivatization resulted in a significant protection, by a factor ranging from 5 to 75, of the internal peptide bond in various N-protected dipeptide amides. These derivatives are readily bioreversible, the conversion to the parent peptide or amino acid amide taking place either by spontaneous hydrolysis at physiological pH, as demonstrated for the N-Mannich bases, or by catalysis by plasma, as for peptidyl--hy-droxyglycine derivatives.     

Synthesis, conformational analysis, and cytotoxicity of conformationally constrained aplidine and tamandarin A analogues incorporating a spirolactam beta-turn mimetic

With the aim of studying the contribution of the beta II turn conformation at the side chain of didemnins to the bioactive conformation responsible for their antitumoral activity, conformationally restricted analogues of aplidine and tamandarin A, where the side chain dipeptide Pro8-N-Me-d-Leu7 is replaced with the spirolactam beta II turn mimetic (5R)-7-[(1R)-1-carbonyl-3-methylbutyl]-6-oxo-1,7-diazaspiro[4.4]nonane, were prepared. Additionally, restricted analogues, where the aplidine (pyruvyl9) or tamandarin A [(S)-Lac9] acyl groups are replaced with the isobutyryl, Boc, and 2-methylacryloyl groups, were also prepared. These structural modifications were detrimental to cytotoxic activity, leading to a decrease of 1-2 orders of magnitude with respect to that exhibited by aplidine and tamandarin A. The conformational analysis of one of these spirolactam aplidine analogues, by NMR and molecular modeling methods, showed that the conformational restriction caused by the spirolactam does not produce significant changes in the overall conformation of aplidine, apart from preferentially stabilizing the trans rotamer at the pyruvyl9-spirolactam amide bond, whereas in aplidine both cis and trans rotamers at the pyruvyl9-Pro8 amide bond are more or less equally stabilized. These results seem to indicate a preference for the cis form at that amide bond in the bioactive conformation of aplidine. The significant influence of this cis/trans isomerism upon the cytotoxicity suggests a possible participation of a peptidylprolyl cis/trans isomerase in the mechanism of action of aplidine.     

Peptides containing the sulfonamide transition-state isostere: synthesis and structure of N-acetyl-tauryl-l-proline methylamide

The structure of the sulfonamide isostere-containing peptide N-acetyl-tauryl-proline methylamide 4 was compared to information on the structure of the peptide N-acetyl-β-alanyl-proline methylamide 6. NMR measurements of the β-alanine containing peptide 6 showed the presence of two conformations due to cis/trans isomerism of the β-Ala-Pro amide bond, whereas the sulfonamide-containing peptide 4 appeared in only one conformation. The crystal structure of N-acetyl-tauryl-proline methylamide 4 gave additional evidence for the absence of cis/trans isomerism. The crystals are orthorhombic, space group P2₁2₁2₁, Z= 4, F(000) = 592, a= 7.5919(3), b= 10.3822(2), c= 17.1908(7) Å, V= 1354.99(8) ų, D_x= 1.359 g cm^?3. The oxygen atoms connected to the sulfur take positions similar to both the cis and trans positions of the carbonyl oxygen of an amide. Consequently the tauryl part is placed perpendicular to the proline α-C-C(O) bond, giving it an extended conformation in contrast to the cis/trans isomers of N-acetyl-β-alanyl-proline methylamide 6. © Munksgaard 1995.     

Statine and its derivatives. Conformational studies using nuclear magnetic resonance spectrometry and energy calculations

The conformations of derivatives of 3(S)-hydroxy-4(S)-amino-6-methylheptanoic acid (statine) and its analogs have been studied by n.m.r. in chloroform and in dimethyl sulfoxide, and by molecular mechanics calculations. The data obtained from these studies indicate that: 1) the coupling constant between NH and C₄H is large, suggesting that the dihedral angle (θ) is near 165° or 0°; 2) the coupling constant between C₄H-C₃H is small, indicating a vicinal bond angle of approximately 90°; 3) the hydrogen deuterium exchange rate of statine amide protons is slow; however, the rate is dependent upon the electron withdrawing substituents adjacent to the amide NH's; 4) intramolecular hydrogen bonds involving the NH of the statine amide group do not stabilize conformations of single amino acid derivatives. Based on the n.m.r. results, four possible conformations of Boc-statine-OMe in solution are possible. MM1 calculations indicate one conformation is especially likely.     

Role of N‐t‐Boc group in helix initiation in a novel tetrapeptide

Abstract: Protecting groups in N‐ and C‐terminal positions play a decisive role in the conformational preference of smaller peptides. Conformational analysis of tetrapeptide derivatives containing Ala, Ile and Gly residues was performed. Peptide 1 , Boc‐Ala‐Ile‐Ile‐Gly‐OMe (Boc: tert‐butyloxycarbonyl) has a predominantly helical turn conformation in all the alcoholic solvents studied, whereas in the solid state it has a β‐sheet conformation. In contrast, peptide 2 , Ac‐Ala‐Ile‐Ile‐Gly‐OMe (Ac: acetyl) has a random coil conformation in solution. The FTIR spectrum of peptide 1 shows a lower frequency of urethane carbonyl, indicating involvement of the carbonyl group in hydrogen bonding in the helical turn.     

Simple and stereospecific homologation of urethane‐protected α‐amino acids to their higher homologs using HBTU1

Abstract: A simple, efficient and stereospecific approach for the homologation of urethane‐protected α‐amino acids to β‐amino acids by the Arndt–Eistert method employing Fmoc‐/Boc‐α‐amino acid and 2‐(1H‐benzotriazole‐1‐yl)‐1,1,3,3‐tetramethyl‐uronium hexafluorophosphate mixture for the acylation of diazomethane synthesizing the key intermediates Fmoc‐/Boc‐α‐aminodiazomethanes as crystalline solids is described.     

A side reaction in solid phase synthesis Insertion of glycine residues into peptide chains via Nim→ Nα transfer

In solid-phase peptide synthesis using N^α-Boc-N^im-tosyl-histidine (Boc-His(Tos)), byproducts having extra Gly residues in the peptide chain were observed at a high rate. When a Boc-amino acid such as Asn was incorporated after assembly of Boc-His(Tos), the N^im-tosyl group was partially or fully cleaved by an activating agent, 1-hydroxybenzotriazole. In the successive coupling reactions, Boc-Gly was incorporated into the free N^im ring as well as the α-amino function, and the N^im-Gly was then transferred to the α-amino group of Gly of the peptide chain after removal of these Boc groups to give extra Gly residues at the position of Gly. This was observed in only the coupling reaction with Boc-Gly and could be circumvented using a more stable N^im protecting group for His, such as a dinitrophenyl group.