Monomer units for the β-bend ribbon structure: MeAib peptides

Monomer units for the fl-bend ribbon smacture: MeAib peptides* V. Moretto, G. Valle, M. Crisma, G. M. Bonora and C. Toniolot Biopolymer Research Centre, CNR, Department of Organic Chemistry, University of Padova, 35131 Padova, Italy

(Received 1 November 1991; revised 15 February 1992) A conformational analysis in CDCI3 solution by using i.r. absorption and 1H nuclear magnetic resonance spectroscopy was performed on the fully blocked dipeptides Z-MeAib-Aib-NHMe, Z-MeAib-L-AIa-NHMe, Z-Aib-MeAib-NHMe, and Z-L-AIa-MeAib-NHMe, representing repeating units of the fl-bend ribbon spiral (an approximate 31o-helix, with an intramolecular H-bonding donor every two residues), where Z represents benzyloxycarbonyl, MeAib ~t-methylaminoisobutyric acid, and NHMe methylamino. The molecular and crystal structures of the first three compounds were also assessed by X-ray diffraction. While the -MeAib-Aib-, -MeAib-L-Ala-, and -Aib-MeAib- sequences give stable fl-bend structures, the preferred conformation of the -L-Ala-MeAib- sequence is open. These results indicate that the MeAib residue is a good fl-bend promoter, but less ej~cient than its unmethylated counterpart at position i + 2. Keywords:~-Methylaminoisobutyricacid;peptides,conformation;1Hnuclearmagneticresonance;i.r. absorption;X-ray diffraction

Introduction The fl-bend ribbon structure, first experimentally verified by Karle et al. 2"3 using X-ray diffraction in the -(Aib-L-Pro)n- sequence, where Aib represents ~-aminoisobutyric acid (Figure 1) of the peptaibol antibiotics~6 zervamicins, may be considered a subtype of the 3 lo-helix (Ref. 7) (typical of Aib-rich peptides s-~°) having approximately the same helical fold of the main chain ( ~ b ~ - 6 0 °, ~ k = - 3 0 °) but whose 1 ~ 4 ( C l o ) intramolecular N-H- • •O = C H-bonding scheme 11 is interrupted by Pro residues at alternate positions. Subsequently, a detailed structural characterization of the fl-bend ribbon spiral has been performed in our laboratory by investigating the complete, terminally blocked, sequential oligopeptide series -(Aib-L-Pro),with n = 1-51'12. This study complemented a series of scattered investigations on shorter -(Aib-L-Pro),oligomers 13-20. More recently, we have extended our analysis of sequences of the type -(Aib-Xxx),-, using the helicogenic ~-hydroxyacid Hib (ct-hydroxyisobutyric acid) to interrupt the backbone H-bonding pattern. Fully protected -(Aib-Hib)~,2-depsipeptides (Figure 1) form the aforementioned ordered secondary structure 2~'22. In this paper we describe a solution and crystal state conformational analysis (by i.r. absorption, ~H nuclear magnetic resonance (n.m.r.) and X-ray diffraction) of two terminally blocked -Aib-MeAib- (Figure 1) and -MeAib-Aib- dipeptides, where MeAib represents ~methylaminoisobutyric acid, which possess the repeating unit of potential fl-bend ribbon structures. In order to assess the helicogenic character of the MeAib residue we have also examined dipeptides where the strong *Part 266 of the Linear Oligopeptides series;for part 265, see ref. 1. tTo whomcorrespondenceshouldbe addressed. 0141-8130/92/040178-07 © 1992Butterworth-HeinemannLimited 178

Int. J. Biol. Macromol., 1992, Vol. 14, August

helix-former Aib 8-1° is replaced by a less efficient Ala residue. Few synthetic and conformational studies have so far dealt with MeAib. The only MeAib-containing peptides synthesized are the dipeptide model compound TosMeAib-Aib-OH (using Tos-MeAib-C1 as the carboxy component)23, where Tos represents p-toluene sulphonyl (tosyl), and inactive [ MeAib ] 1-analogues of angiotensin II (using N-unprotected MeAib on Merrifield solid phase resin) zn-z6. In all cases the MeAib residue is located at the N-terminus of the peptide chain. The rate of cyclization of the hydantoic acid from MeAib is 105 times faster than that of the Gly analogue zT, an unequivocal manifestation of the gem-dimethyl or Thorpe-Ingold

I

CH3

,H 3

-~Aib-- Hib~

(~H3

-~ NH--C --CO--O I

C

I

CH 3

CH 3

~

CH 3

H3

CO-~n

I

I

I

I

CH 3

CH 3

CH 3

Figure 1 A schematic representation of the monomer units for the fl-bend ribbon structure investigated to date

MeAib peptides: V. Moretto et al. effect2s. The i.r. absorption spectrum of Ac-MeAibNHMe in a dilute CC14 solution z9 is indicative Of the occurrence of the intramolecularly H-bonded CT-folded conformer11 to a small extent.

Experimental Synthesis of peptides H-MeAib-OH was purchased from Sigma, PyBroP (bromo-tris-pyrrolidino-phosphonium hexafluorophosphate) from Novabiochem and EDC (N-ethyl, N'-(3dimethylaminopropyl)-carbodiimide) from Fluka. ZAib-OH 23'3°, (Z-L-AIa)2 O, and Z-L-Ala-NHMe were prepared according to published procedures. Removal of the Z-protecting group was achieved by hydrogenolysis in methanol with 10% Pd/C as catalyst. Z-MeAib-OH. This compound was prepared from H-MeAib-OH and Z-C1 in a 2M NaOH-acetone mixture: yield 83%; m.p. 136-137°C (from ethyl acetate-petroleum ether); thin-layer chromatography (t.l.c.) (silica-gel plates 60F-254, Merck) Rfl (chloroform-ethanol 9:1) 0.60, Rf2 (1-butanol-acetic acidwater 3:1 : 1 ) 0.80. I.r. absorption (KBr) Vmax 3091, 1734, 1654 cm- 1.1H_n.m.r. (CDC13) 6 7.34 (m, 5H, Z phenyl), 5.13 (s, 2H, Z CH2), 2.98 (s, 3H, MeAib N-CH3), 1.49 (s, 6H, MeAib flCH 3). (Z-MeAib)20. This compound was prepared either from Z-MeAib-OH and 0.5 equivalents of thionyl chloride in anhydrous ethyl acetate in the presence of triethylamine or from Z-MeAib-OH and 0.5 equivalents of EDC hydrochloride in anhydrous acetonitrile: yield 70-85%; oil; Rfl 0.90. I.r. absorption (film between KBr discs) Vmax 1823, 1778, 1749, 1694cm -1. XH-n.m.r. (CDCI3) ~ 7.33 (m, 10H, 2 Z phenyl), 5.11 (s, 4H, 2 Z CH 2), 2.89 (s, 6H, 2 MeAib N-CH3) , 1.42 (s, 12H, 2 MeAib flCH3 ). Z-MeAib-NHMe. This compound was prepared from Z-MeAib-OH and 35% aqueous triethylamine in an acetronitrile-tetrahydrofuran 1:1 mixture in the presence of EDC hydrochloride: yield 68 %; oil; Rfl 0.75; Rf2 0.85. I.r. absorption (film) Vm,x 3348, 1700, 1663, 1537 cm- 1.1H_n.m.r. (CDC13) ~ 7.34 (m, 5H, Z phenyl ), 5.69 (broad m, 1H, methylamino NH), 5.11 (s, 2H, Z CHz), 2.99 (s, 3H, MeAib N-CH3), 2.65 (d, 3H, methylamino CH 3), 1.45 (s, 6H, MeAib flCH 3). Z-Aib-NHMe. This compound was prepared from Z-Aib-OH as described above for Z-MeAib-NHMe: yield 62%; m.p. 105-106°C (from ethyl acetate-petroleum ether); Rfl 0.60; Rf2 0.85. I.r. absorption (KBr) vm~x 3416, 3349, 1690, 1672, 1530cm -1. 1H-n.m.r. (CDC13) 6 7.34 (m, 5H, Z phenyl), 6.31 (broad m, 1H, methylamino NH), 5.28 (s, 1H, Aib NH), 2.80 (d, 3H, methylamino CH 3), 1.58 (s, 6H, Aib f l c n 3). Z-MeAib-Aib-NHMe (1). This compound was prepared by reacting (Z-MeAib)20 with H-Aib-NHMe 31 in anhydrous acetronitrile under reflux for 72 h: yield 17%, m.p. 185-186°C (from ethyl acetate-petroleum ether); Rfl 0.40; Rfz 0.85. I.r. absorption (KBr) Vm,x 3369, 3331, 1689, 1648, 1541, 1519cm -1. 1H-n.m.r. (CDC13) 6 7.33 (m, 5H, Z phenyl), 7.18 (broad m, 1H, methylamino NH), 5.73 (s, IH, Aib NH), 5.14 (s, 2H,

Z CH2), 3.01 (s, 3H, MeAib N-CH3), 2.70 (d, 3H, methylamino CH3), 1.43 and 1.41 (2s, 12H, Aib and MeAib f l f H 3 ).

Z-MeAib-I.-Ala-NHMe (2). This compound was prepared by reacting (Z-MeAib)2 ° with H-L-Ala-NHMe in anhydrous acetonitrile at room temperature overnight: yield 32%, m.p. 149-150°C (from ethyl acetatepetroleum ether); R~I 0.65; Rf2 0.85; [ ~ ] ~ ° = - 3 . 0 ° (c = 0.5, methanol). I.r. absorption (KBr) Vm~x 3352, 3294, 1689, 1651, 1554, 1520cm -1. 1H-n.rn.r. (CDC13) 7.33 (m, 5H, Z phenyl), 7.04 (broad m, 1H, methylamino NH), 5.84 (d, 1H, Ala NH), 5.12 (m, 2H, Z CH2), 4.40 (m, 1H, Ala ~CH), 3,01 (s, 3H, MeAib N - C H 3 ), 2.70 (d, 3H, methylamino CH 3 ), 1.47 and 1.46 (2s, 6H, MeAib flCH3), 1.35 (d, 3H, Ala flcn3). Z-Aib-MeAib-NHMe (3). This compound was prepared by reaction of Z-Aib-OH with H-MeAib-NHMe in the presence of PyBroP 32'33 and diisopropylethylamine in anhydrous methylene chloride at room temperature for 2 weeks: yield 3%, m.p. 177-178°C (from ethyl acetate-petroleum ether); Rfl 0.80; Rfz 0.95. I.r. absorption (KBr) Vmax 3376, 3256, 1701, 1675, 1626, 1535 cm- 1.1H.n.m.r. (CDCI3) fi 7.34 (m, 5H, Z phenyl), 6.82 (broad m, 1H, methylamino NH), 5.11 (s, 2H, Z CHz), 5.07 (s, 1H, Aib NH), 2.88 (s, 3H, MeAib N - C H 3), 2.75 (d, 3H, methylamino CH 3 ), 1.48 and 1.32 (2s, 12H, Aib and MeAib flcn3). Z-L-Ala-MeAib-NHMe (4). This compound was prepared by reacting (Z-L-AIa)20 with H-MeAib-NHMe in the presence of 4-dimethylaminopyridine in anhydrous acetonitrile under reflux for 7 days. The crude product, obtained after the usual work-up, largely contaminated by the unreacted symmetrical anhydride, was purified by flash-chromatography on silica gel with a chloroformethanol 9:1 mixture as eluent: yield 4%; m.p. 181-182°C (from ethyl acetate-petroleum ether); Rfl 0.70; Rf2 0.80. I.r. absorption (KBr) VmaX3307, 1692, 1650, 1538 cm -1. 1H-n.m.r. (CDC13) 6 7.34 (m, 5H, Z phenyl), 5.69 (broad m, 1H, methylamino NH), 5.62 (d, 1H, Ala NH), 5.08 (s, 2H, Z CH2), 4.62 (m, 1H, Ala c~CH), 3.04 (s, 3H, MeAib N-CH3), 2.75 (d, 6H, methylamino CH3) , 1.46 and 1.45 (2s, 6H, MeAib f l f n 3 ), 1.32 (d, 3H, Ala flCH3 ). Infrared absorption Infrared absorption spectra were recorded by using a Perkin Elmer Model 580-B spectrophotometer equipped with a Perkin Elmer 3600 infrared data station. The band positions are accurate to +_1 cm-1. Cells with pathlengths of 0.1, 1.0 and 10 mm (with CaF z windows) were used. Spectrograde deuterochloroform (99.8% d) was purchased from Merck. For the solid-state measurements the KBr disc technique was used. 1H nuclear magnetic resonance The 1H-n.m.r. spectra were recorded with a Bruker Model AM-400 spectrometer. Measurements were carried out in deuterochloroform (99.96% d, Merck) and dimethyl-d6 sulphoxide (99.96% d6, Fluka) with tetramethylsilane as the internal standard. X-ray diffraction Intensities were collected on a Philips PW 1100 four-circle diffractometer operating in the 0/20 scan

Int. J. Biol. Macromol., 1992, Vol. 14, August

179

MeAib peptides: V. Moretto et al.

Table 1 Crystallographic data for the three MeAib-containing peptides (1-3) Parameter

Z-MeAib-Aib-NHMe (1)

Z-MeAib-L-Ala-NHMe (2)

Z-Aib-MeAib-NHMe (3)

Mol. formula Mol. weight (a.m.u.) Crystal dimensions (mm) Crystal system Space group Z (molecules/unit cell) a (A) b (A) c (A) V (A 3) d (calculated) (gcm- 3) d (experimental) (g cm- 3) F(100) # (cm- 1) No. of unique reflections Reflections with F i> 7a(F) Final R value Rw W Temperature (K) Crystallization solvent" Residual electron density in final AF map (e A -a) S

C18H27N30 4

C17H25NaO4

C18H27N304

349.4 0.1 x 0.4 x 0.6 Orthorhombic P212t21 4 18.816 (2) 10.955 (2) 9.049 (2) 1865.6 (6) 1.24 1.23 188 0.53 2549 991 0.038 0.040 1/[a2(F) + 0.0012F2] 298 MeOH/H20 0.14

335.4 0.4 × 0.4 × 0.5 Orthorhombic P212121 4 18.412 (2) 10.410 (2) 9.431 (2) 1807.6 (6) 1.23 1.23 180 0.53 2462 1489 0.058 0.067 1/[tr2(F) + 0.0027F2] 298 MeOH/H20 0.2•

349.4 0.25 × 0.25 × 0.30 Orthorhombic P212121 4 21.714 (2) 10.956 (2) 8.374 (1) 1992.2 (5) 1.17 1.17 188 0.50 2022 712 0.078 0.083 1/[a2(F) + 0.009F2] 298 AcOEt/EP 0.27

1.07

1.45

1.01

"MeOH, methanol; AcOEt, ethyl acetate; EP, petroleum ether

mode to 20 = 56 ° with graphite-monochromatized MoK~ radiation (2 = 0.7107 A). A total of 2549 unique reflections for 1, 2462 for 2, and 2022 for 3 were measured, of which 991, 1489, and 712, respectively, had intensities F>~7a(F). During data collection three standard reflections were measured every 180min to check stability of the crystal and the electronics. Intensities were corrected for Lorentz and polarization factors. No absorption correction was applied. The three structures were solved by direct methods using SHELX 8634 and refined by blocked least-squares methods with anisotropic thermal parameters for all non-hydrogen atoms. For 1 and 2 hydrogen atoms were localized on the AF map and isotopically refined; the hydrogen atoms of 3 were partially localized on the AF map and not refined. All calculations were performed on the IBM 370/158 computer of the University of Padova using the SHELX 7635 program. The atomic scattering factors for all atomic species were calculated according to Cromer and Waber s6. Crystallographic data for the three structures are reported in Table 1. Tables of final atomic coordinates, and lists of bond distances, bond angles, and torsion angles have been deposited and are available from the Cambridge Crystallographic Data Centre.

Results and discussion Solution conformational analysis Information on the solution preferred conformations of the MeAib-containing N~-protected dipeptide methylamides 1-4 was obtained in a solvent of low polarity (CDC13) by i.r. absorption and ~H-n.m.r. The i.r. absorption spectra in the informative N - H stretching region (amide A) are illustrated in Figure 2 and the most relevant ~H-n.m.r. data are shown in Figure 3.

180

Int. J. Biol. Macromol., 1992, Vol. 14, August

The i.r. absorption curves, which remain essentially unchanged in the 0.1-10 mM concentration range, are characterized by the following bands: (i) 3485-3483 c m - 1 and 3465-3455cm -1, assigned to free methylamino N - H groups29; (ii) 3437-3426 cm -1, assigned to free peptide and urethane N - H groupslS'29'37; and (iii) 3386-3370cm -~, assigned to intramolecularly Hbonded methylamino N - H groups 29'37. These assignments are corroborated by the observation that the two bands above 3450 cm-1 occur also in Z-MeAib-NHMe (spectrum not shown) and Ac-MeAib-NHMe 29. From an analysis of the i.r. absorption spectra it turns out that intramolecularly H-bonded folded forms are extensively adopted by peptides 1-3, whereas there is no evidence for their occurrence in Z-L-Ala-MeAib-NHMe

(4). The delineation of the inaccessible (or intramolecularly H-bonded) NH group in CDC13 by ~H n.m.r, was performed using solvent (Me2SO) dependency of N H chemical shifts 38. Assignment of NH resonances has been carried out by analysis of chemical shifts (resonances of urethane NH groups are typically seen at higher fields than amide or peptide N H groups15'37), peak multiplicities, and homonuclear spin decoupling experiments. There is no evidence from the 1H-n.m.r. spectra of minor conformations, suggesting that tertiary urethane and peptide bonds occur only in the trans disposition in solution. The effect of peptide concentration (in the range 10 1.0 mM) on NH chemical shifts is negligible. Figure 3 shows that in the absence of self-association two classes of NH protons are observed. The former class includes protons whose chemical shifts are almost insensitive to the addition of the strong H-bonding acceptor solvent Me2SO 39 (methylamino NH protons of peptides 1-3); the latter class includes all other protons, displaying a behaviour characteristic of unshielded protons (remarkable sensitivity of chemical shifts to solvent composition).

MeAib peptides: V. Moretto et al.

3500

3450

3400

3350

3000

3450

WAVENUMBER

3400

3350

3300

3250

( c m -1)

Figure 2 Infrared absorption spectra in the N-H stretching region of (A) Z-MeAib-Aib-NHMe (1), (B) Z-MeAib-L-Ala-NHMe (2), (C) Z-Aib-MeAib-NHMe (3), and (D) Z-L-Ala-MeAib-NHMe (4) in CDC13 solution (conc. 1.0 mM)

b

8.0 I- a

tc

t°

NHMe

NHMe

NHMe

7.0 E GL

NHMe Aib

6.0

Aib

]fJ~

Ala

5.0 3

6

9

3

6

9

3

6

9

3

6

9

%Me2SO in CDCI 3

Figure 3 Plot of NH chemical shifts in the 1H-n.m.r. spectra of (a) Z-MeAib-Aib-NHMe (1), (b) Z-MeAib-L-AIa-NHMe (2), (c) Z-Aib-MeAib-NHMe (3), and (d) Z-L-Ala-MeAib-NHMe (4) versus increasing percentages of Me2SO added to the CDC13 solution (v/v). Peptide concentration: 1.5 mM In summary, these 1H-n.m.r. data support the conclusion that in CDC13 solution the methylamino NH protons of peptides 1-3 are involved in an intramolecular H-bond; in contrast, the corresponding proton of peptide 4 is free. The occurrence of the signal of the methylamino NH proton of peptide 4 at significantly higher fields compared with the corresponding protons of peptides 1-3 represents an additional proof in favour of our conformational assignments. These, more detailed, conclusions agree well with the indications extracted from the i.r. absorption investigation.

Crystal-state conformational analysis The molecular structures of peptides 1-3 with numbering of atoms are illustrated in Figures 4-6,

respectively. Table 2 lists the torsion angles of the peptide backbone and the N-terminal protecting group. Since peptides 1 and 3 are achiral, helices of both screw senses concomitantly occur in their crystals. For an easier comparison with chiral peptide 2, only the right-handed helices ofpeptides 1 and 3 will be discussed. The geometry of N H . . . O = C intra- and intermolecular H-bonds is reported in Table 3. The structures of the three peptides are similar. Peptides 1 and 3 adopt a regular type-III fl-bend 11'4°'41 (the building unit of the polypeptide 3it-helix 7 ), while a slightly distorted type-Ill//-bend (intermediate between type-Ill and type-I fl-bends) is seen for peptide 2. In particular,the sets of qS,~ktortion angles 42 of the MeAib residues in the three peptides fall in the 'helical' region

Int. J. Biol. Macromol., 1992, Vol. 14, August

181

M e A i b peptides." V. Moretto et al.

I'

C~=

•.~

C1

Ou

a

NI

Ou

c(7)

c(71

c(1) -

~ O i \ ) c~ I

~c c16)

/oo

NT /

//

Oo

Ct*

ctl) 1 c(2)

(

(si

c (3)( 02

O?

Figure 4 Molecular structure of Z-MeAib-Aib-NHMe (1) with numbering of the atoms. The intramolecular H-bond is indicated as a broken line

c~'

c;

co)

(3~2

c(4)

C~

C~

c(3)~ c(s)

01

c(4)

c;(

c:d 11 IP O=

Figure 5 Molecular structure of Z-MeAib-L-Ala-NHMe (2) with numbering of the atoms. The intramolecutar H-bond is indicated as a broken line

Figure 6 Molecular structure of Z-Aib-MeAib-NHMe (3) with numbering of the atoms. The intramolecular H-bond is indicated as a broken line

A of the conformational map 43, their average values being - 5 0 . 5 °, - 4 1 . 8 °, respectively. It seems therefore that an -(Aib-MeAib),- sequential peptide will give rise to a more regular fl-bend ribbon than that exhibited by an -(Aib-L-Pro).- peptide L12. In the three peptides the intramolecular H-bond is seen between the amide NT-H aand urethane O o = C o groups. The N . - - O separations are in the range 2.960(5)-3.013(11 ) A 4¢-46. All urethane, peptide and amide bonds are in the usual trans arrangement 47, with only those between MeAib and Aib residues (~ol torsion angles) of peptides 1 and 2 deviating remarkablyfrom planarity. The Z-urethane moiety of the three peptides is of the common b-type 4s, both 01 and 090 torsion angles being trans. The 02 and 03 torsion angles, giving the orientation of the phenyl ring relative to the urethane moiety, have values in the ranges 76.6-81.7 ° and 24.2-69.5 °, respectively. Interestingly, in the observed distribution of 02 in crystalline Z-derivatives, the values are concentrated in three regions, close to 90 °, - 9 0 °, and 180 °, respectively; conversely, the distribution of 03 is broad, extending over its entire range 4s.

Table 2 Relevant torsion angles (deg)= for the three MeAib-containing peptides (1 3)

0a 02 01 090 • 1 W1 o91 (1)2 W2 fox

Angle

Z-MeAib-Aib-NHMe (1)

Z-MeAib-L-AIa-NHMe (2)

Z-Aib-MeAib-NHMe (3)

C(2)-C(1)-C(7)-Ou C(1)-C(7)-O,-C'o C(7)-Ou-Co-N1 O,-Co-NI-C~ Co-NI-C'~-C'~ N1-C~-C'I-N2 C1-C1-N2-C2 C'~-N2-C~-C'2 N2-C~-C2-NT C~-C2-Nr-CT

34.3 (7) 76.6 (6) 179.4 (5) -175.1 (4) -52.9 (5) -42.9 (6) -170.5 (4) -63.3 (6) -28.0 (6) --174.5 (5)

24.2 (8) 78.6 (6) -177.4 (4) -174.2 (4) -51.2 (5) -43.0 (5) -172.5 (4) -78.8 (5) -10.1 (6) --179.8 (5)

69.5 (11) 81.7 (10) -170.4 (8) 174.5 (7) -46.9 (12) -40.5 (13) 179.7 (9) -47.4 (13) -39.6 (13) 178.2 (9)

"Estimated standard deviation values are given in parentheses

182

Int. J. Biol. Macromol., 1992, Vol. 14, August

MeAib peptides: V. Moretto et al. Table 3 Geometry of the intra- and intermolecular H-bonds in the crystals of the three MeAib-containing peptides ( 1 - 3 ) Distance (A) D o n o r ( D ) Acceptor(A)

Z-MeAib-Aib-NHMe (1) NT--HT N2-H2 Z-MeAib-L-Ala-NHMe (2) NT--HT N2-H 2 Z-Aib-MeAib-NHMe (3) NT--HT N1-H 1

Symmetry equiv. of A

D...A

H...A

D--H .... A

Oo 02

x,y,z - x - l , y + 1/2,-z-1/2

3.000(6) 3.048(5)

2.136(70) 2.325(48)

158.0(60) 160.7(47)

O0 0 2

x,y,z - x , y - I / 2 , - z + 1/2

2.960(5) 2.961(5)

2.229(38) 2.096(55)

157.9(36) 152.9(48)

O0 O1

x,y,z - x , y - 1 / 2 , - z + 1/2

3.013(11) 2.783(10)

2.134(7) 1.752(7)

133.6(5) 160.8(5)

In the crystals of peptides 1 and 2, chains of molecule are generated along the v direction through (peptide) N2-H2 • • •O2=C~ (amide) intermolecular H-bonds. On the other hand, the molecules of peptide 3 are held together along the same direction by a N - H . . - O = C intermolecular H-bond between (urethane) N1-HI and O1=C] (peptide) groups. The N . . . O distances are in the range 2.783( 10 )-3.048(5 ) A**-.6.

16

Conclusions

19

We have synthesized four MeAib-containing peptides, including for the first time two peptides where the MeAib residue is not located at the N-terminal position of the amino acid sequence. A solution and crystal-state conformational analysis showed that -MeAib-Aib- and -Aib-MeAib- dipeptide sequences form stable type-III fl-bend structures, suggesting that they may be useful monomer units for the formation of a regular //-bend ribbon spiral. A comparison with dipeptides containing the -MeAib-L,Ala- and -L-Ala-MeAib, sequences, which indicated that the former is folded in a//-bend structure while the latter is open, and with literature data on -Aib-L-Ala-, -L-Ala-Aib -49-53, and -L-AIa-L-Ala-54'55 sequences, is strongly in favour of the conclusion that the MeAib residue, although being a fl-bend promoter better than L-Ala at position i + 1, is less efficient than Aib, its unmethylated analogue, at position i + 2.

20

References 1 2 3 4 5 6 7 8 9 10 11 12 13

Crisma, M., Anzolin, M., Bonora, G. M., Toniolo, C., Benedetti, E., Di Blasio, B. et al. Gazz. Chim. Ital. (in press). Karle, I. L., Flippen-Anderson, J., Sukumar, M. and Balaram, P. Proc. Natl Acad. Sci. USA 1987, 84, 5087 Karle, I. L., Flippen-Anderson, J., Agarwalla, S. and Balaram, P. Proc. Natl Acad. Sci. USA 1991, 88, 5307 Benedetti, E., Bavoso, A., Di Blasio, B., Pavone, V., Pedone, C., Toniolo, C. and Bonora, G. M. Proc. Natl Acad. Sci. USA 1982, 79, 7951 Brfickner, H. and Graf, H. Experientia 1983, 39, 528 Mathew, M. K. and Balaram, P. Mol. Cell. Biochem. 1983, 50, 47 Toniolo, C. and Benedetti, E. TIBS 1991, 16, 350 Toniolo, C. and Benedetti, E. ISIAtlas o f Science. Biochemistry 1988, 1, 225 Toniolo, C. and Benedetti, E. Macromolecules 1991, 24, 4004 Karle, I. L. and Balaram, P. Biochemistry 1990, 29, 6747 Toniolo, C. CRC Crit. Rev. Biochem. 1980, 9, 1 Di Blasio, B., Pavone, V., Saviano, M., Lombardi, A., Nastri, F., Pedone, C. et al. J. Am. Chem. Soc. (in press) Venkataram Prasad, B.V., Shamala, N., Nagaraj, R., Chandrasekaran, R. and Balaram, P. Biopolymers 1979,18,1635

14 15

17 18

21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Angle (deg)

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