J. Peptide Res. 49, 1997, 556-562 Printed in UK all rights reserved

Copyright 0 Munksgaard 1997 JOlJRNAL OF PEPTIDE RESEARCH

ISSN 1397-VV2X

Aza-peptides 11. X-Ray structures of aza-alanine and aza-asparagine-containing peptides

FREDERIC ANDRk ', GUY BOUSSARD ', DANIEL BAYEUL ', CLPUDE DIDIERJEAN *, ANDRE AUBRY and MICHEL MARRAUD

' LCPM, CNRS- URA-494, ENSIC-INPL, Nuncy, Frunce and ' LCM3B, CNRS- LIRA-801. UHP, Vandeuvre les Nuticj,, France

Received 16 October, revised 7 December 1996, accepted for publication 8 February 1997

In order to determine the structural consequences of the N"/C'H exchange in aza-peptides, we have solved the crystal molecular structures of some derivatives containing the aza-analogue of asparagine [Z-AzAsn( Me)-NMe, ( 1 ), Z-AzAsn( Me)-Pro-NHiPr (2) and Piv-Pro-AzAsn( Me)-NHiPr (5)], aspartic acid [Z-AzAsp(0Et)-Pro-NHiPr ( 3 ) and alanine ( Boc-AzAla-Pro-NHiPr (4)], by using X-ray diffraction. They reveal that the r-nitrogen accommodates a pyramidal (1-4) or planar (5) structure depending on the sequence. When pyramidal, the r-nitrogen assumes the R (D-like) chirality. All of the derivatives but 1 adopt either a &folded (2-4) or /&-folded (5) structure in which the (AzAsn)NSH bond is intramolecularly hydrogen-bonded to the r-nitrogen. Q Munksgaard 1997.

Key words: aza-alanine; aza-asparagine: aza-peptides; crystal molecular structure; pseudopeptide; X-ray diffraction

Aza-peptides, in which nitrogen has been substituted for at least one of the CH" groups, constitute a pseudopeptide series with an cw-modification allowing retention of the side chains. Aza-analogues have been really studied since the early sixties and actively developed by Gante ( I ) and by Dutta and Morley (2). ha-residues have been incorporated in eledoisin analogues ( 3), in various angiotensin converting enzyme (4) and renin (5, 6) inhibitors. More recently, their synthesis has been particularly investigated by Gante (7, 8) and by us (9, 10) in liquid-phase peptide synthesis, and by Gray et al. ( 1 1 ) in solid-phase peptide synthesis. In the following, the aza-analogue of an amino acid residue Xaa will be denoted as AzXaa.

The three-dimensional data on aza-peptides are relatively limited (5, 7, 10, 12-16). In previous studies we showed that the aza-analogue of proline exhibits very particular conformational properties: unlike Pro, it induces P-folding of the Ala-&Pro sequence, but an open structure of the AzPro-Ala sequence (10, 13-15). Although they are linked to a carbonyl, each of the nitrogen atoms in the AzPro unit adopts clearly a non-planar structure, probably because of sterical hindrances due to the nearly planar structure of the five-membered pyrazolidine ring. On the contrary, the (AzA1a)a-nitrogen is nearly planar in the crystal structure of Piv-Pro-AzAla-NHiPr (9), and the same

556

holds true for the AzPhe and AzIle residues in an all-aza-analogue of a renin inhibitor (7).

We have solved the crystal molecular structures of the five AzAla- and AzAsx-containing aza-peptides 1-5 (Asx denoting Asp or Asn) in order to evaluate the structural impact of the Na/CaH exchange, and more especially, the behaviour of the carboxamide side chain in the AzAsn-containing derivatives. In four of them, the aza-residue is preceded or followed by proline which is known to influence strongly on the folding tendencies of the peptide chains (17). We will also refer to the crystal structure of the AzAla- containing aza-peptide 6 (9). 1 Z-AzAsn( Me)-NMe, 2 Z-AzAsn( Me)-Pro-NHiPr 3 Z-AzAsp(0Et)-Pro-NHiPr 4 Boc-AzAla-Pro-NHiPr 5 Piv-Pro-AzAsn (Me)-NHiPr 6 Piv-Pro-AzAla-NHiPr (9)

X-RAY DIFFRACTION

The synthesis of the AzAla and AzAsx derivatives are reported in the preceding paper in this series (18). Single crystals of 1 were grown by slow evaporation of a iPr,O/THF (9/1) solution, and single crystals of 2-5 from a iPr,O/AcOEt (9/1) mixture. The X-ray diffraction data were collected on a MACH 3 four- circle diffractometer, equipped with a rotating anode,

Aza-peptides: X-ray structure

TABLE 2 Out-of-plane deviation (A) of the a-nitrogen atom, as defined by the three atoms bonded to it, in the molecular structures of six crystullirrd

aza-pep tides

in the o/2@-scan mode. The independent reflections were measured at room temperature in theo 1-70 O 0- range using Cu-Ka radiation (A = 1.541 8 A) mono- chromatized by a graphite crystal. During data collec- tion, two standard reflections were measured every 2 h in order to check the crystal stability. Intensities were corrected for Lorentz and polarization effects, but no absorption correction was applied. The main crystallographic data are given in Table 1. The crystal structures were solved by direct methods making use of SHELXS (19). During SHELXL refinement (20), heavy atoms were affected by anisotropic thermal factors while hydrogen atoms were located on E-map differepes and affected by an isotropic thermal factor of 5 A2. The residual R-factors are indicated in Table 1.

RESULTS

Geometry of the aza-residue The six crystal structures reveal the possibility for the a-nitrogen to adopt either a planar (two cases) or non-planar (four cases) structure, although it is linked to a carbonyl. In derivatives 5 and 6 the deviation of the a-nitrogen out of the plane, defined bx the three atoms bonded to it, is only 0.08 and 0.04 A, respect- ively, wh$reas 1-4 exhibit a deviation in the 0.25-0.32 A range (Table 2), which may be cpmpared with that for the AzPro residueo (0.3-0.4 A range) (15), and with the value of 0.45 A corresponding to a standard sp3 nitrogen. This difference results in two sets of bond angles around the a-nitrogen, whereas the other dimensions are not significantly affected (Fig. 1). One notes also that the dimensions of the AzAsn side chain do not differ from those of the Asn residue (2 1 ) .

Z-AzAsn( Me)-NMe, Boc- AzAla-Pro-NHiPr Z-AzAsp (0Et)-Pro-NHiPr Z- AzAsn ( Me)-Pro-NHiPr Piv-Pro-AzAsn (Me)-NHiPr Piv-Pro-AzAla-NHiPr"

0.25 0.32 0.28 0.26 0.08 0.04

a Ref. 9

I H

CP

H FIGURE 1 Average bond lengths (a) and bond angles (b) of the aza-residue main chain. The upper and lower bond angle values refer to a planar and pyramidal a-nitrogen, respectively.

TABLE 1 Crystal data and structure reJinement (SHELX programs)

Z-AzAsn( Me)- Boc-AzAla- Z-AzAsp(0Et)- Z-AzAsn( Me)- Piv-Pro- NMe, Pro-NHiPr Pro-NHiPr Pro-NHiPr AzAsn( Me)-NHiPr

Crystal system Space group Unit cell dimensions

a, A b, A c, A lt deg z Density (calculated), g/cm3 Reflections collected Independent reflections [ I> 2a(I)] Final R-indices Rl [ I > 2 m 1 wR, [Z>2a(I)] R, (all data) wR, (all data)

Monoclinic

18.459( 4) 7.976( 2)

22.552(4) 106.7(2)

8 1.288

3068 2887

0.055 0.154 0.076 0.206

Orthorhombic p212121

9.715( 10) 10.993 (2) 17.01 3 (3) 90

4 1.201

2018 1862

0.038 0.097 0.052 0.126

Orthorhombic p21212,

12.460(4) 13.268( 1 ) 14.024( 1 ) 90 4 1.216

2433 202 I

0.039 0.108 0.053 0.146

Orthorhombic p21212,

8.852( 1) 14.000(6) 17.942( 2) 90 4 1.256

2400 1781

0.047 0.136 0.071 0.182

Orthorhombic p212121

7.399 (2) 8.129(2)

35.270(6) 90

4 1.157

2372 2337

0.061 0.170 0.077 0.246

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F. Andre et al.

The amide preceding the a-nitrogen is classically trans planar, but the ?mide bond C'-N is a little longer by about 0.03 A than that of the standard peptide bond (22). The greatest differences from the standard peptide group logically concern the bond lengths and bond angles around the r-nitrogen: (i) thF N-N" and W-CB bonds are shorter by about 0.06 A than their homologous boads in peptides; (ii) the Na-C' bond length of 1.39 A exceeds the dimension of a standard amide bond, but is shorter by about

0.13 A than the homologous c"-C' bond in peptides; (iii) the N-Na-C' angle is larger by about 4-5 O than the N-C-C' bond angle.

Crystal molecular structures The molecules of compound 1 adopt two mirror- image T-shape conformations with a non-planar a-nitrogen (Table 2). Stereoviews of the S (L-like) molecule are represented in Fig. 2 illustrating that the side chain and the C-terminus are in a nearly planar

FIGURE 2 Stereoviews of the crystal molecular structure of Z-AzAsn( Me)-NMe,, 1, with the S (L-like) chirality.

TABLE 3 Main torsional angles in tlie crystal molecular Structures of rlre AzAla- or AzAsx-containing aza-peptides'

Z-AzAsn( Me)- Z-AzAsn( Me)- Z-AzAsp(0Et)- Boc-AzAla- Piv-Pro- Piv-Pro- NMe,b Pro-NHiPr Pro-NHiPr Pro-NHiPr AzAsn (Me)-NHiPr AzAla-NHiPr"

w

4 * x' xZ x3 x4

'4' '*' 'w' ' X I ' '%2'

Y'

0

-178.0(2) 175.2(3)

-75.7(4) - 15.0(3) - 170.6(4) - 28.9(4)

38.8(5)

15.3(4) -32.8(5)

-61.2(3) - 51.7( 5) - 37.8( 3) - 35.6(5)

68.1(2) 77.0(4) - 164.2(2) 8.6(5)

179.0(2)/-10.6(3) -178.2(3)

-175.0(2) -175.8(4)

Z/Boc/Piv

Pro - 66.9( 3)

- 178.8(2)

-23.9(4) - 178.4(3) - 34.2( 3)

40.3(4) - 30.6(4)

9.4( 3)

-61.4( 3) - 29.6( 3)

AzAlafAzAsx

-179.9(2) 76.3(3)

- 161.4(2) - 176.8(3)

172.3 (2)

- 66.7( 3) - 17.7(4) 172.0(3) - 34.4( 3 )

41.4(3) -31.5(3)

10.1(3)

- 58.1 (3) -24.7( 3) 175.2(2)

177.7(4)

- 62.3( 5 ) 140.1(3)

31.4(6)

38.3(8)

- 178.0(3)

-42.9(7)

-19.7(9)

81.1 (4) 2.0(4)

104.2( 3 ) -178.8(4)

- 17.8(4) - 177.1 (3)

176.6(4)

- 55.4(4) 120.9(3) 179.7(2) 30.0(5)

38.9(5) -30.8(4)

- 2 2 4 5)

89.3 (4) 17.8(5)

168.9(2)

The aza-residue torsional angles are defined exactly as for the amino acid residue (23). The angular values reported here refer to the molecule with a R (D-like) r-nitrogen. Ref. 9.

558

Aza-peptides: X-ray structure

Nu is, in that compound, practically planar (Table 2). Note that, contrary to 2, (AzAsn)N6H is not inter- molecularly hydrogen-bonded in 5 in favour of the resident intramolecular hydrogen bond (AzAsn)N'H-..O = C(Pro), closing the eight- membered chelation cycle, identical to a Gly-like 'hydrazino turn' ( 16).

extended conformation, practically perpendicular to the extended N-terminal fragment. It is interesting to note that the 4 and $ values (Table 3) are not those generally favored in the case of proteinogenic residues of the same chirality, for which opposite values are rather expected (22). Each molecule is connected to four neighbors by a network of intermolecular N-H . . .O = C hydrogen bonds (Table 4).

The three derivatives 2-4 having an AzXaa-Pro sequence share the same structure (Fig. 3) folded by a hydrogen bond of the i + 3 -ti type between the (iPr)NH and (Boc/Z)CO sites (Table 4). All the amide bonds are trans planar, and the values of the torsional angles, listed in Table 3, are typical of a PI- turn. In all of the three cases, the a-nitrogen of each aza-residue is far from planarity (Table 2), and assumes the R (d ike) chirality. The AzAsn side chain in 2 is folded over the peptide backbone (Fig. 3a) so as the (AzAsn)NsH proton not only stands at a hydrogen-bond distance from (AzAsn)N" forming a nearly planar five-membered pseudocycle, but also is involved in an intermolecular interaction (Table 4). The absence of the (AzAsn)NsH group in 3 results in an extended [AzAsp(OEt)] side chain. All of these molecules in the crystal are intermolecularly hydrogen-bonded to two (3 and 4) or three (2) neighbouring molecules.

The derivative 5 with the Pro-AzAsn sequence adopts a PII-type structure (Fig. 4) folded by a (iPr)NH to (Piv)CO interaction. The AzAsn side chain is again folded over the peptide backbone in such a way that the (AzAsn)N'H group interacts with (Pro)CO, and once again holds at a hydrogen- bond distance from (AzAsn)N" (Table 4), although

DISCUSSION The N"/C"H exchange, generating aza-residues, results in huge conformational changes which were not expected for such an isoelectronic modification, apparently rather tiny. The first consequence is the a-nitrogen pyramidal state, which depends on the sequence. When not planar and belonging to an aza- residue preceding the chiral L-proline (derivatives 2- 4), it assumes the R (D-like) chirality. Whatever be its planar or pyramidal nature, it can be involved in a nearly planar five-membered pseudocycle closed by a distorted (AzAsn)N'-H..-N" side-chain-main- chain hydrogen bond. This suggests the existence of some residual electronic charge on the a-nitrogen, in good agreement with the increased length of the N"-CO bond, thus highlighting a less sp2-hybridized character.

The aza-analogue of either Ala or Asp or Asn residue induces a &folded turn, even when followed by proline, which is known to favour rather /?-folded Pro-Xaa sequences ( 17). It is likely that this particular behaviour comes from the possibility for the a-nitro- gen to accommodate a d i k e chirality. Actually, the D-Ala-L-Pro sequence is PI, '-folded in both solution and crystalline state, whereas the L-Ala-L-Pro

TABLE 4 Dimensions (A". ") of the inter- and intramolecular hydrogen bonds in the crystal structures of aza-peptides 1-5

Donor Acceptor Symmetry code N...O/N

(AzAsn)NH (AzAsn)N8H

(iPr)NH (AzAsn)N'H ( AzAsn)NH ( AzAsn)N6H

(iPr)NH ( AzAsn)NH

(iPr)NH (AzA1a)NH

(iPr)NH ( AzAsn)N6H ( AzAsn)N6H ( AzAsn)NH

( AzAsn)CYO ( AzAsn)CO

(Z)CO ( AzAsn)N" (Pro)CO (Pro)CO

(Z)CO (Pro)CO

(Z)CO (AzA1a)CO

(Piv)CO (AzAsn)N" (Pro)CO ( AzAsn)CYO

Z-AzAsn(Me)-NMe, (1) - X, Y, 312 - z 2.871(2) 1 / 2 - ~ , 1/2+y, 312-2 2.959(2)

x, Y, z 3.078(5) x, Y, 2 2.773(5) -x, -0.5+y, 0.5-z 2.828(5) -x, - 0 . 5 + ~ , 0.5-2 2.963(5)

x, Y, z 3.143(4) - 0 . 5 + ~ , 0.5-y, 2-2 2.925(3)

x, Y3 z 3.037(4) -x, 0.5+y, -0.5-2 2.868(3)

x, Y. z 3.055 (4) x, Y, z 2.789(4) x, Y, z 2.924( 4) -x, 0.5+,~, 0.5-z 2.770(4)

Z-AzAsn( Me)-Pro-NHiPr (2)

Z-AzAsp(0Et)-Pro-NHiPr (3)

Boc-AzAla-Pro-NHiPr (4)

Piv-Pro-AzAsn (Me)-NHiPr ( 5 )

1.85(2) 1.95(1)

2.10(1) 2.38(3) 1.83( 3) 2.10(3)

2.19(3) 1.94(3)

2.04( 3) 1.86(2)

2.15(3) 2.43(5) 1.94(3) 1.80(4)

174(2) 171(2)

160(3) lOl(2) 1 W 2 ) 140(3)

157(3) 161(4)

166(3) 169(3)

147(3) 99(3)

160(3) 157(4)

F. AndrC et al.

A

FIGURE 3 Stereoviews of the &folded crystal molecular structures of Z-AzAsn( Me)-Pro-NHiPr, 2 (a). Z-AzAsp(0Et)-Pro-NHiPr, 3 (b ) and Boc- AzAla-Pro-NHiPr, 4 (c). The intramolecular hydrogen bonds are indicated by broken lines.

560

Aza-peptides: X-ray structure

n

U

FIGURE 4 Stereoviews of the /?,,-folded crystal molecular structure of Piv-Pro-AzAsn(Me)-NHiPr, 5. The intramolecular hydrogen bonds are indicated by broken lines.

sequence assumes an open structure under the same conditions (24-27).

The aza-asparagine residue induces structural prop- erties and side-chain to main-chain interactions different from those of asparagine (28). We have shown that Asn is often involved in a particular arrangement, called Asx-turn, where the CyO car- bony1 of the main-chain Asn (i) is hydrogen-bonded to the amide NH in position i+2 (29). The Asx- turn, particularly favoured by the Asn-Pro sequence, is not observed in 2. On the other hand, the BI-turn in the Pro-Asn sequence, which is stabilized by the intramolecular (Asn)NH to (Asn)CYO interaction (21), turns into a &turn in the Pro-AzAsn sequence. In fact, the Asn side chain essentially acts as a proton acceptor with its (Asn)CYO carbonyl, but the AzAsn side chain operates as a proton donor by means of its (AzAsn)N'H group (Table 4; Fig. 4).

Whether the AzXaa residue in 1-6 is located in position i+ 1 of a p,-turn or i+2 of a jII-turn, it adopts two quasi-mirror image conformations (Table 3 ) corresponding to the average torsional angles 4, $ = - 60 O , - 30 O (compounds 1-4) and 70°, 10" (compounds 5 and 6). It is interesting to note that AzAla behaves similarly to AzAsn, indicating that the carboxamide group in the latter is not responsible for the different backbone conformational properties of the homologous peptides and aza- peptides. This peculiarity should confer on the aza- residues some interesting properties for the induction of fl-folded structures.

REFERENCES

1. Gante, J. (1989) Synthesis, 405-413 and references cited therein 2. Dutta, A.S. & Morley, J.S. (1975) J. Chem. Soc., Perkin Trans.

1, 1712-1720

3. Niedrich, H & Oehme, C. (1972) J. Prakt. Chem. 314,759-768 4. Greenlee, W.J., Thorsett, E.D., Springer, J.P., Patchett, A.A.,

Ulm, E.H. & Vassil, T.C. (1984) Biochem. Biophys. Res. Commun. 122, 791-797

5. Gante, J. & Kahlenberg, H. (1991) Chem. Z. 115, 215-217 6. Gante, J., Krug, M., Lauterbach, G., Weitzel, R. & Hiller, W.

(1995) J Peptide Sci. 2, 201-206 7. Gante, J., Krug, M., Lauterbach, G. & Weitzel, R. (1994) in

Hodges, R.S. & Smith, J.A., eds., Peptides: Chemistry and Biology, Proceedings of the 13th American Peptide Symposium, pp. 299-301, ESCOM, Leiden, The Netherlands

8. Gante, J. (1994) Angew. Chem., Int. Ed. Engl. 33, 1704-1712 9. Benatalah, Z., Aubry, A,, Boussard, G. & Marraud, M. (1991)

Int. J. Peptide Protein Res. 38, 603-605 10. Lecoq, A,, Boussard, G., Marraud, M. & Aubry, A. (1991)

Tetrahedron Lett. 33, 5209-521 2 11. Gray, C.J., Quibell, M., Beggett, N. & Hammerk, T. (1992)

Int. J. Peptide Protein Res. 40, 351-362 12. Pinnen, F., Luisi, G. & Lucente, G. (1993) J. Chem. Soc..

Perkin Trans. 1, 819-824 13. Lecoq, A,, Boussard, G., Marraud, M. & Aubry, A. (1993)

in Schneider, C.H.& Eberle, A.N., eds., Peptides 1992. Proceedings of the 22th European Peptide Symposium, pp. 601-2, ESCOM, Leiden, The Netherlands

14. Zouikri, M., Boussard, G., Marraud, M., Didierjean, C., Del Duca, V. & Aubry, A. (1995) in Maia H.L.S., ed., Peptides 1994, Proceedings of the 23th European Peptide Symposium, pp. 507-8, Escom, Leiden, The Netherlands

15. Lecoq, A., Boussard, G., Marraud, M. & Aubry, A. (1993) Biopolymers 33, 1051-1059

16. Marraud, M. & Aubry, A. (1996) Biopolymers (Peptide Sci.)

17. Mac Arthur, M.W. & Thornton, J. (1991) J. Mol. Biol. 218,

18. Andre, F., Didierjean, C., Boussard, G., Vanderesse, R., Aubry, A. & Marraud, M. (1996) in Kaumaya P.T.P. & Hodges R.S. eds., Peptides: Chemistry, Structure and Biology, Proceedings of the 14th American Peptide Symposium, pp. 703-704, Mayflower Scientific Ltd., UK

19. Sheldrick, G.M. (1990), Acta Crystallogr., Sect. A 46,467-473

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40, 45-83

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20. Sheldrick, G.M. (1993), SHELXL 93, Program for the Refinement of Crystal Structures. University of Gottingen, Germany

21. Mcharfi, M., Aubry, A,, Boussard, G. & Marraud, M. (1986) Eur. Biophys. J. 14, 43-51

22. Benedetti, E. (1977) in Goodman, M. & Meienhofer, J., eds., Peptides, pp. 251-213, Wiley, New York

23. IUPAC-IUB Commission on Biochemical Nomenclature (1985) J. Biol. Chem. 260, 16-42

24. Aubry, A,, Protas, J., Boussard, G. & Marraud, M. (1979) Acta Crystallogr.. Sect. B 35, 694-699

25. Aubry, A., Protas, J., Boussard, G. & Marraud, M. (1980) Acta Crystallogr., Sect. B 36, 321-326

26. Aubry, A,, Protas, J., Boussard, G. & Marraud, M. (1980) Acta Crystallogr., Sect. B 36, 2825-2827

27. Vitoux, B., Aubry, A,, Cung, M., T. & Marraud, M. (1986) Int. J. Peptide Protein Res. 21, 617-632

28. Srinivasan, N., Anuradha, V.S., Ramakrishnan, C., Sowahamini, R. & Balaram, P. (1994) Int. J. Peptide Protein Res. 44, 112-122

29. Abbadi, A,, Mcharfi, M., Aubry, A,, Premilat, S., Boussard, G. & Marraud, M. (1991) J. Am. Chem. SOC. 113, 2729-2735

Address:

Dr. Guy C. Boussard LCPM-ENSIC, B. P. 451 54 001 Nancy Cedex France

Tel: 03 83 17 51 96 Fax: 03 83 31 99 77 E-mail: boussard@lcpm.ensic.u-nancy.fr

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