PREFERRED CONFORMATION OF THE tert-BUTOXYCARBONYLAMINO GROUP IN PEPTIDES

Structural parameters, derived from X-ray crystallographic data, have been compiled for amino acid and linear peptide derivatives which contain the N-terminal tert-butoxycarbonyl (Boc) group or its next higher homolog, the tert-amyloxycarbonyl group. The comparison of the geometry of the urethane group in Boc-derivatives with that of the peptide group shows small differences in bond angles about the trigonal carbon, because of altered interactions when a CαH group of a peptide unit is replaced by an ester oxygen. In contrast to the strong preference of the peptide bond for the trans form (except when it precedes proline), the urethane amide bond adopts both the cis and trans conformations in crystals. The cis urethane conformation is preferred in crystals of compounds with a tertiary nitrogen (such as Boc-Pro) or in structures stabilized by strong intermolecular interactions. Conformational energy computations on Boc-amino acid N'-methylamides indicate that the trans and cis conformations of the urethane amide bond have nearly equal energies (even for amino acids other than proline), in contrast to the peptide bond, for which the trans conformation has a much lower energy. The computed increase of the cis content in Boc-amino acid derivatives (as compared with the corresponding N-acetyl derivatives) is consistent with the observed distributions of conformations in crystal structures and with n.m.r. studies in solution. Usually, the substitution of a Boc for an N-acetyl end group does not alter the conformational preferences (as indicated by φ, Ψ values and relative energies) of the amino acid residue which follows the end group when the amide bond is trans. Particular conformations, however, can be stabilized by strong attractive interactions between some side chains (e.g. that of phenylalanine) and the bulky Boc end group.     

Context‐dependent conformation of diethylglycine residues in peptides

Abstract: Diethylglycine (Deg) residues incorporated into peptides can stabilize fully extended (C₅) or helical conformations. The conformations of three tetrapeptides Boc‐Xxx‐Deg‐Xxx‐Deg‐OMe (Xxx = Gly, GD4; Leu, LD4 and Pro, PD4) have been investigated by NMR. In the Gly and Leu peptides, NOE data suggest that the local conformations at the Deg residues are fully extended. Low temperature coefficients for the Deg(2) and Deg(4) NH groups are consistent with their inaccessibility to solvent, in a C₅ conformation. NMR evidence supports a folded β‐turn conformation involving Deg(2)‐Gly(3), stabilized by a 4 → 1 intramolecular hydrogen bond between Pro(1) CO and Deg(4) NH in the proline containing peptide (PD4). The crystal structure of GD4 reveals a hydrated multiple turn conformation with Gly(1)–Deg(2) adopting a distorted type II/II′ conformation, while the Deg(2)–Pro(3) segment adopts a type III/III′ structure. A lone water molecule is inserted into the potential 4 → 1 hydrogen bond of the Gly(1)–Deg(2) β‐turn.     

Linear tripeptide conformation

The structures of two tripeptides, Cbz-glycylglycyltyrosine methyl ester (ZGGYOMe) and Cbz-glycyl-(D,L)tyrosylglycine ethyl ester (ZGYGOEt) have been determined from single-crystal X-ray diffraction data. Crystals of ZGGYOMe are monoclinic, space group P2₁, with a= 12.427(3), b= 4.999(3), c= 17.401(6) Å, β= 99.98(2)° and Z= 2. The final R-index is 0.049 for 1698 reflections with I≥2 σ(I). Crystals of ZGYGOEt are monoclinic, space group P2₁/n with a= 12.134(8), b= 14.614(3), c= 26.154(9) Å, β= 98.78(4)°, Z= 8. The final R-index is 0.067 for 4457 reflections with I≥2 σ(I). Both peptides adopt highly extended structures; principal torsion angles are ω₀= 175.0(4)°, φ₁= 69.2(5)°, ψ₁=? 154.9(4)°, ω₁=?175.8(4)°, φ₂= 165.4(4)°, ψ₂= 154.2(3)°, ω₂= 169.6(3)°, φ₃=?94.8(5)°, ψ₃=?47.6(5)° for ZGGYOMe and, for the two independent molecules of ZGYGOEt, ω₀= 177.9(4)°, 178.9(4)°, φ₁=?172.0(4)°, 169.7(4)°ψ₁= 174.4(4)°, ?162.5(4)°; ω₁= -170.1(4)°, 176.7(4)°; φ₂=?130.8(4)°, 130.3(5)°; ψ₂= 162.8(4)°, ?163.3(4)°; ω₂=?177.6(4)°, 176.2(4)°; φ₃=? 169.9(4)°, 172.9(4)°; ψ₃=? 168.2(4)°, 160.9(4)°. The structures are of interest since the first one adopts a conformation unlike those of related GGX sequences and the latter shows an antiparallel hydrogen-bonding pattern.     

Aromatic interactions in tryptophan‐containing peptides: crystal structures of model tryptophan peptides and phenylalanine analogs*

Abstract: The crystal structures of the peptides, Boc‐Leu‐Trp‐Val‐OMe ( 1) , Ac‐Leu‐Trp‐Val‐OMe ( 2a and 2b), Boc‐Leu‐Phe‐Val‐OMe ( 3 ), Ac‐Leu‐Phe‐Val‐OMe ( 4 ), and Boc‐Ala‐Aib‐Leu‐Trp‐Val‐OMe ( 5 ) have been determined by X‐ray diffraction in order to explore the nature of interactions between aromatic rings, specifically the indole side chain of Trp residues. Peptide 1 adopts a type I β‐turn conformation stabilized by an intramolecular 4→1 hydrogen bond. Molecules of 1 pack into helical columns stabilized by two intermolecular hydrogen bonds, Leu(1)NH…O(2)Trp(2) and IndoleNH…O(1)Leu(1). The superhelical columns further pack into the tetragonal space group P4₃ by means of a continuous network of indole–indole interactions. Peptide 2 crystallizes in two polymorphic forms, P2₁ ( 2a ) and P2₁2₁2₁ ( 2b ). In both forms, the peptide backbone is extended, with antiparallel β‐sheet association being observed in crystals. Extended strand conformations and antiparallel β‐sheet formation are also observed in the Phe‐containing analogs, Boc‐Leu‐Phe‐Val‐OMe ( 3 ) and Ac‐Leu‐Phe‐Val‐OMe ( 4 ). Peptide 5 forms a short stretch of 3₁₀‐helix. Analysis of aromatic–aromatic and aromatic–amide interactions in the structures of peptides, 1 , 2a , 2b are reported along with the examples of 14 Trp‐containing peptides from the Cambridge Crystallographic Database. The results suggest that there is no dramatic preference for a preferred orientation of two proximal indole rings. In Trp‐containing peptides specific orientations of the indole ring, with respect to the preceding and succeeding peptide units, appear to be preferred in β‐turns and extended structures.     

Immunoreactivity and conformation of the P-P-G-M-R-P-P repetitive epitope of the Sm autoantigen

Anti-Sm antibodies are usually considered highly specific for systemic lupus erythematosus (SLE), while anti-UI RNP antibodies are found in high titers in patients with mixed connective tissue disease (MCTD). The sequence P¹-P-G-M-R-P-P⁷, present in three copies in the Sm (Ul-U6 RNA-protein complex) autoantigen, is an important functional domain of the antigenic determinants. The immunoreactivity of this proline-rich repetitive epitope was investigated by testing sera with various autoantibody specificities for reactivity against this epitope, as well as its conformational properties by means of ID and 2D ¹HNMR spectroscopy. It was found that the P-P-G-M-R-P-P epitope is recognized mainly by anti-UlRNP and/or anti-Sm positive sera, but also by anti-Ro(SSA) (hY1RNA-protein complex) and anti-La(SSB) (hY1RNA-protein complex) positive sera, although these sera are negative for anti-UlRNP and anti-Sm. Conformational analysis of the proline-rich epitope in DMSO-d₆ solution obtained from lyophilized aqueous solution at pH 5 showed the presence of at least three conformers. The main conformer A (62%) is stabilized by an ionic interaction between the guanidinium and the C-terminal carboxylate groups, and the Pro⁶-Pro⁷ peptide bond adopts the cis form. A type II β-turn is also present in the N-terminal sequence (Pro¹-Pro-Gly-Met⁴-) of this conformer. Conformer B (21%) is also stabilized by a similar ionic interaction, as in conformer A, while the NMR data indicate the absence of a folded structure in the N-terminal tetrapeptide of this conformer. Conformer C (17%) adopts a completely extended structure. The multiple conformers of the P-P-G-M-R-P-P may offer some explanation for the reactivity of sera with various autoantibody specificities against this epitope. © Munksgaard 1996.     

Conformational properties of aminosuccinyl peptides.

The synthesis and crystal structure of Boc-L-aminosuccinyl-glycine methyl ester is reported. The crystal structure was solved by direct methods and refined to a final R value of 0.064 for the 761 observed reflections (I ≥ 2.5 σ(I)). The molecule is highly extended and the succinimide ring adopts a puckered conformation intermediate between that of a “pure envelope” and a “pure twist” conformation. The molecule was also studied by empirical potential energy calculations. One of the minima of the potential energy map closely corresponds to a type II‘β reverse turn. The results are discussed in connection with the stereochemical constraints imposed by the presence of a cyclic imide structure to the conformational freedom of a polypeptide chain.     

Structure and conformation of linear peptides

The crystal structure of t-Boc-glycyl-L-phenylalanine (C₁₄H₂₂N₂O₅, molecular weight = 298) has been determined. Crystals are monoclinic, space group P2₁, with a = 7.599(1)Å, b = 9.576(2), c = 12.841(2), β = 97.21(1)°, Z = 2, D_m = 1.149, D° = 1.168 g · cm^-3. Trial structure was obtained by direct methods and refined to a final R-index of 0.064 for 1465 reflections with I> 1s?. The peptide unit is trans planar and is nearly perpendicular to the plane containing the urethane moiety. The plane of the carboxyl group makes a dihedral angle of 16.0° with the peptide unit. The backbone torison angles are ω₀ = - 176.9°, ø₁ = - 88.0°, ψ₁ = - 14.5°, ω1 = 176.4°, ø₂ = - 164.7° and ψ2 = 170.3°. The phenylalanine side chain conformation is represented by the torsion angles χ₁ = 52.0°, χ₂ = 85.8°.     

Structure and conformation of linear peptides

The crystal structure of a dipeptide tert-butyloxycarbonyl-l -alanylglycine monohydrate (C₁₀H₁₈N₂O₅·H₂O), molecular weight 264, has been determined. The crystals are monoclinic, space group P2₁, with a= 10.767(1), b= 6.317(1), c= 10.981(2) Å, β= 109.15(2)°, and Z= 2, D_c= 1.24 g cm^?3. The structure was solved by direct methods and refined to 3 final R-index of 0.045 for 856 reflections (sin θ/λ < 0.55 Å^?1) with I > 2 σ. The N-terminus of the molecule blocked with the t-Boc group is uncharged and the C-terminus exists in an unionized state. The peptide unit is trans and shows slight deviations from planarity. (Δω= 3.1°). The peptide backbone is folded, with torsion angles of φ₁= -76.0(5), ψ₁= 164.3(4), ω₁= 176.9(5), φ₂= 116.1(5), ψ₂₁= - 2.8(7) and ψ₂₂= 177.8(4)°. The conformation about the urethane bond (C5–N1) is trans. The urethane group is essentially planar. The conformation of the boc group is trans–trans.     

Acyclic peptides as conformational models

The tripeptide Boc-Aib-Leu-Pro-NHMe crystallizes in the orthorhombic space group P2₁2₁2₁ with a = 9.542, b = 15.200, c = 18.256 Å and Z = 4. Each peptide is associated wth two water molecules in the asymmetric unit of the crystal. The structure has been solved by direct methods and refined to an R-value of 0.069. The peptide adopts a structure without any intramolecular hydrogen bond. The three residues occupy distinctly different regions of the Ramachandran map: Aib in the left-handed 3₁₀-helical region (± = 67°, ± = 23°), Leu in the β-sheet region (± = - 133°, ± = 142°) and Pro in the poly (Pro) II region (± = - 69°, ± = 151°). An interesting observation is that each water molecule participates in four hydrogen bonds with distorted tetrahedral coordination about the oxygen atom.     

Secondary structure formation in N-substituted peptides*

A systematic conformational analysis on several model peptides with N-substituted amino acids was performed on the basis of ab initio MO theory at the HF/6-31G* and HF/3-21G levels with inclusion of solvation effects to study the influence of N-substitution on the formation of typically secondary structural elements, e.g. β sheets, helices and turns. The conformational flexibility of some structures was examined by means of molecular dynamics simulations in the gas phase and in solution. The results show a restriction of the conformational flexibility of the peptide chain after introduction of an N-substituted amino acid, N-substitution makes β sheet formation more difficult. Several consecutive N-substituted amino acids in a sequence lead to conformers different from those found on the energy hypersurface of the corresponding N-unsubstituted peptides. There is a strong tendency to form periodically helical conformations, e.g. the polyglycine II or the α helix, which can be extended over several N-substituted amino acid residues. As long as 1 → 4 hydrogen bond formation remains possible, the major types of β turns can be formed with a distinct preference for the βII and βIa turns. The β turn in particular is considerably destabilized.     

Structure and conformation of linear peptides

The tripeptide, L-prolyl-glycyl-glycine, crystallizes in the trigonal space group P3₂, with a = b = 8.682(2)Å, c= 12.008(2) and Z = 3. The structure was solved by direct methods and refined to an R-value of 0.07 for 727 reflections (I > 1.0s?). The molecule exists as a zwitterion in the crystal. The peptide units and trans and show significant deviations from planarity (ω₁ = 169.7^o, ω₂=-170.1^o). The peptide backbone adopts a left-handed helical conformation similar to that of polyglycine II and polyproline II.     

Structure and conformation of peptides containing the sulphonamide junction

N-acetyl-tauryl-l -phenylalanine methyl ester 1 has been synthesized. The crystal structure and molecular conformation of 1 have been determined. Crystals are monoclinic, space group P2₁ with a = 5.088(2), b = 17.112(17), c = 9.581(6) Å, β= 92.34(4)^M-0, Z = 2. The structure has been solved by direct methods and refined to R = 0.043 for 2279 reflections with I < 1.5σ(I). The sulphonamide junction maintains the peptide backbone folded with Tau and Phe C^α atoms in a cisoidal arrangement, the torsion angle around the S-N bond being 65.4^M-0. In this conformation the p-orbital of the sulphonamide nitrogen lies in the region of the plane bisecting the O-S-O angle, thus favouring dα-pα interactions between nitrogen and sulphur atoms. The S-N bond with a length of 1.618 Å has significant α-bond character. The CO-NH is planar and adopts trans conformation. The Tau residue is extended with the Tau-C^α₁-C^β_a bond anti-periplanar to the S-N bond. The Phe side chain conformation corresponds to the statistically most favoured g^- rotamer and exhibits a χ¹ torsion angle of –67.5^M-0. The packing is characterized by intermolecular H-bonds which the Tau and Phe NH groups form with the acetyl carbonyl and one of the two sulphonamide oxygens, respectively.     

Design of peptides with αβ-dehydro residues: Synthesis,crystal structure and molecular conformation of N-Boc-L-Ile-ΔPhe-L-Trp-OCH3

The dehydro-peptide Boc-L-Ile-ΔPhe-L-Trp-OCH₃ was synthesized by the azlactone method in the solution phase. The peptide was crystallized from methanol in an orthorhombic space group P2₁2₁2₁ with a = 10.777(2), b= 11.224(2), c= 26.627(10) Å. The structure was determined by direct methods and refined to an R value of 0.069 for 3093 observed reflections [l≥ 2σ(l)].The peptide failed to adopt a folded conformation with backbone torsion angles: φ₁, = 90.8(8)°, ψ₁= -151.6(6)°, φ₂= 89.0(8)°, ψ₂= 15.9(9)°, φ₃= 165.7(7)°, ψ^T₃= -166.0(7)°. A general rule derived from earlier studies indicates that a three-peptide unit sequence with a ΔPhe at the (i+ 2) position adopts a β-turn II conformation. Because the branched β-carbon residues such as valine and isoleucine have strong conformational preferences, they combine with the ΔPhe residue differently to generate a unique set of conformations in such peptides. The presence of β-branched residues simultaneously at both (i+ 1) and (i+ 3) positions induces unfolded conformations in tetrapeptides, but a β-branched residue substituted only at (i+ 3) positron can not prevent the formation of a folded β-turn II conformation. On the other hand, the present structure shows that a β-branched residue substituted at the (i+ 1) position prevents the formation of a β-turn II conformation. These observations indicate that a β-branched residue at the (i+ 1) position prevents a folded conformation whereas it cannot generate the same degree of effect from the (i+ 3) position. This may be because of the trans disposition of the planar ΔPhe side-chain with respect to the C=O group in the residue. The molecules are packed in an anti-parallel manner to generate N₂-H₂…O₂ (-x,y-1/2, -z+ 3/2) and N^ε1₃-H^ε1₃…O₁(-x,y -1/2, -z+ 3/2) hydrogen bonds.     

Structure and conformation of linear peptides

The tripeptide, glycyl-glycyl-L-isoleucine, crystallizes as a monohydrate in the monoclinic space group P2₁, with a = 12.746(2), b = 6.172(1), c = 8.643(1) Å, β = 99.77(2)°, and Z = 2. The structure was solved by direct methods and refined to an R-value of 0.039 for 917 (I > 1°) reflections. The molecule exists as a zwitterion in the crystal. The peptide units are trans and show significant deviations from planarity. The plane of peptide units and of the carboxyl group are nearly mutually perpendicular to each other. The peptide backbone torsion angles are: ø₁ = - 171.2°, ω₁ = - 176.8°, ø₂ = - 106.1°, ø₂ = - 150.7°, ø₂ = - 172.1°, ø₃ = - 70.9°, ø₃ = 136.5°. For the side-chain of isoleucine, ø₁ = - 58.1°, ø₂ = 169.7° and the system of bonds C′-C^α-C^β-C γ₁^-Cδ is trans zig-zag. The packing arrangement involves spatial segregation of polar and nonpolar moieties.     

Conformational influences of N2-methylation in chemotactic peptides

Physical and structural data on two of a series of related N-formyl methionine peptides are reported. The effects of peptide bond N-methylation on chemotactic response and on conformational properties observed in solution, in the solid state and by conformational energy calculations are described.     

Structure and conformation of linear peptides

L-tyrosyl-L-tyrosine crystallizes as a dihydrate in the orthorhombic system, space group C222₁, with a = 12.105(2), b = 12.789(2), c = 24.492(3) Å, Z = 8. The structure was solved by direct methods and refined to a final R-value of 0.059 for 1740 observed reflections. The molecule exists as a zwitterion, the peptide unit is trans planar, and the backbone torsion angles correspond to an extended conformation, with e₁ = 149.4°, e₂ = - 161.2°, e₂ = 158.3°. The values of the side-chain torsion angles (χ₁, χ₂) are (- 58.8°, - 63.1°) for the first tyrosine and (- 171.7°, - 116.5°) for the second. The planes of the aromatic rings are nearly parallel (dihedral angle of 6.1°), and their centers are separated by 10.9 Å. The carboxyl plane forms a dihedral angle of 23.8° with the plane of the peptide bond.     

Design of peptides: synthesis,crystal structure and molecular conformation of N-Boc-L-Val-δPhe-L-Ile-OCH3

The dehydro-peptide Boc-L-Val-δPhe-L-Ile-OCH₃ was synthesized by the azlactone method in the solution phase. The peptide crystallized from a methanol/dimethyl sulfoxide (95:5) mixture in space group P6₁, with a=b= 15.312(1), c= 22.164(5) Å. The structure was determined by direct methods and refined to an R value of 0.098 for 1589 observed reflections [I≥ 1.5 σ(I)]. The peptide adopts an S-shaped conformation with torsion angles: ø₁=-127(1), ψ₁= -44(1), ø₂, = 67(1), ψ₂, = 37(1), ø₃,=-82(1)°. The side-chain torsion angles in δPhe of X¹₂= 1(2), X^2.1₂= 7(2) and X_2.22 = 177(1)° indicate that the δPhe residue is essentially planar. In valyl residue the two side-chain torsion angles are X¹₁= -65(1) and X²₁= 177(1), whereas the torsion angles in Ile are X^1,1₃= 72(2), X^1,2₃= -159(2), X²₃= 150(2)°. This is the first peptide which does not adopt a folded conformation for a sequence with a δPhe at the (i+ 2) position. The molecular packing in the crystals is stabilized by several hydrogen bonds: N₁-H₁?O₁’= 2.77(1) Å, N₂-H₂?O₁’= 2.95(1) Å, N₃-H₃?O₂=2.85(1) Å and a possible weak interaction N₂-H₂?O₁’3.29(1) Å- within the columns of molecules along the c-axis and van der Waals forces between the columns. © Munksgaard 1996.     

Structure and conformation of peptides involving prolyl residue

The dipeptide, L-prolyl-L-leucine monohydrate (C₁₁ H₂₀ N₂ O₃· H₂O, molecular weight, 246.3) crystallizes in the monoclinic space group P2_1/, with cell constants: a = 6.492(2)Å b = 5.417(8)Å c = 20.491(5)Å, β= 96.59(2)°, Z = 2, Do = 1.15g/cm³, and Dc = 1.142g/cm³. The structure was solved by SHELX-86 and refined by full matrix least squares methods to a final R-factor of 0.081 for 660 unique reflections (I > 2σ (I)) measured on an Enraf Nonius CAD-4 diffractometer (CuK_x, λ= 1.5418 4AR, T = 293 K). The peptide linkage exists in the trans conformation. The pyrrolidine ring exists in the envelope conformation. The values of the sidechain torsion angles are: ψ₁= -59.3(13)°, ψ₂₁= -63.1(16)° and ψ₂₂= 174.8(15)° for leucine (C-terminal). The crystal structure is stabilised by a three-dimensional network of N—H… O, O—H… O, and C—H…O hydrogen bonds.     

Crystal structure,conformation, and potential energy calculations the chemotactic peptide N- formyl-l-Met-l-Leu-l-Phe-OMe

The tripeptide N-formyl-L-Met-l -Leu-l -Phe-OMe (FMLP-OMe) crystallizes in the orthorhombic system, space group P 2₁2_l2₁, with the following unit-cell parameters: a = 21.727, b = 21.836, c = 5.133Å, Z = 4. The structure has been solved and refined to a final R of 0.068 for 1838 independent reflexions with I > 2σ(I). The peptide backbone is folded at the Leu residue (φ_L=?67.7,Ψ_L=?49.1°) without intramolecular hydrogen bonds. Considering each peptide plane, the Leu side-chain is oriented on the same side of that of the Phe residue and on the opposite side of that of the Met residue, respectively. The crystal conformation differs from all the other conformations proposed for FMLP-OMe and the anionic form of N-formyl-l -Met-l -Leu-l -Phe-OH (FMLP) in solution accounts for the amphiphilic character of the peptide, giving rise, through intermolecular hydrogen bonds, to a stacking of molecules which could be maintained in the aggregation states experimentally observed in solvents of low polarity. Intramolecular potential energy calculations have ben carried out in order to compare the energies of the various backbone conformers.     

Structure and conformation of linear peptides

The tripeptide, glycyl-glycyl-L-valine, crystallizes as a dihydrate in the monoclinic space group P₂1, with a = 5.786(1), b = 7.954(2), c = 14.420(3)A, β= 93.85(2)d?, Z = 2. The structure was solved by direct methods and refined to an R-value of 0.040 for 876 observed reflections. The molecule exists as a zwitterion in the crystal. The peptide planes show significant deviations from planarity. The chain conformation resembles a reverse turn if the orientation of the carboxyl group is also taken into account. An intramolecular water bridge links the amino and carboxyl ends of the molecule. The crystal packing involves spatial segregation of polar and nonpolar moieties.