8898
J. Am. Chem. SOC.1993, 115. 8898-8906
Conformational Perturbations Induced by N-Amination and N-Hydroxylation of Peptided Virginie Dupont,? Alain L e ~ o q , ”Jean-Paul ~ Mangeot,t Andre Aubryf Guy Boussard,? and Michel Marraud’vt Contribution from the Laboratory of Macromolecular Physical Chemistry, CNRS- URA-494, ENSIC-INPL, BP 451, 54001 Nancy Cedex, France, and Laboratory of Mineralogy and Crystallography, CNRS- URA-809, University of Nancy I, BP 239, 54506 Vandoeuvre Cedex, France Received February 19, I 9 9 P
Abstract: Amination and hydroxylation of the amide nitrogen in a peptide chain have little influence on the local
geometry, but both affect the hydrogen-bonding network, and therefore the conformational properties of the modified peptide. An experimental study in solution (IR spectroscopy and ‘H-NMR) and in the solid state (X-ray diffraction) has been carried out on the N-amino and N-hydroxy analogues of the two RCO-Pro-NHMe and RCO-Pro-Gly-NHiPr peptides known to adopt preferentially the y- and @-turnstructures, respectively. The N-amino group is a weak proton donor which does not interact significantly with the peptide chain. On the contrary, the N-hydroxyl group is a strong proton donor giving close contacts with the peptide carbonyls. The resulting folded conformers of an expanded y- or ,%like type, presenting an 8- or 1I-membered cycle instead of a 7- or 10-membered cycle in the cognate peptides have been also analyzed by a SYBYL molecular dynamics simulation.
The studies of structureactivity relationships for biologically active peptides are largely based on peptide analogues with geometry constrained by covalent cycles or modified side Various cyclic structures designed to mimic the @- and y-turns have also been proposed.l’-16 Another means to influence the conformational properties of a peptide is to modify the peptide bond i t ~ e l f . 2 Besides J ~ ~ ~ the probable dropping of biodegradation kinetics, this change should perturb the intramolecular hydrogent Laboratory of Macromolecular Physical Chemistry, ENSIC-INPL. 1 Presentaddress:
DIEP, CENdeSaclay, 91 191Gif/YvetteCedex, France. Laboratory of Mineralogy and Crystallography, University of Nancy I. 11 Abbreviations: Bzl, benzyl; Boc, tert-butyloxycarbonyl; DCCI, dicyclohexylcarbodiimide; DMAP, 4-(dimethylamino)pyridine; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; Fmoc, 9-fluorenylmethyloxycarbonyl; NMM, N-methylmorpholine, OSu, N-oxysuccinimide; Piv, pivalyl; THF, tetrahydrofuran; Z, benzyloxycarbonyl. a Abstract published in Advance ACS Abstracts, September 1, 1993. (1) Hruby, V. J. Life Sci. 1982, 31, 189-199. (2) Spatola,A. F. In ChemistryandBiochemistryofAminoAcids, Peptides and Proteins; Weinstein, B., Ed.; Marcel Dekker: New York, 1983; Vol. 7, pp 267-351. (3) Kessler, H. In Trends in Drug Research; Claasen, V., Ed.; Elsevier: Amsterdam, 1990; pp 73-84. (4) Hruby, V. J.; Kazmierski, W.; Kawasaki, A. M.; Matsunaga, T. 0.In Peptide Pharmaceuticals; Ward, D. J., Ed.; Open University Press: Milton Keynes, England, 1990; pp 135-184. (5) Hruby, V. J.; AI-Obeidi, F.; Kazmierski, W. Biochem. J. 1990, 268, 249-262. (6) Marshall, G. R.; Motoc, I. In Molecular Graphics and Drug Design; Burgen, A. S . W., Roberts, G. C. K., Tute, M. S., Eds.; Elsevier: Amsterdam, 1986; pp 115-156. (7) Hruby, V. J.; Sharma,S. D. Curr. Opin. Biotechnol. 1991,2,599-605. ( 8 ) Marshall, G. R. Tetrahedron 1993, 49, 3547-3558. (9) Toniolo, C. Int. J. Pept. Protein Res. 1990, 35, 287-300. (10) Osano, Y. T.; Nagai, U.; Matsuzaki, T. Anal. Sci. 1989,5,625-626. (1 1) Saragovi, H. I.; Fitzpatrick, D.; Raktabutr, A,; Nakanishi, H.; Kahn, M.; Greene, M. I. Science 1991, 253, 792-794. (12) Genin. M. J.; Gleason, W. B.; Johnson, R. J. J. Org. Chem. 1993,58, 86&866. (13) Baldwin, J. E.;Hulme,C.; Edwards, A. J.; Schofield, C. J. Tetrahedron Lett. 1993, 34, 1665-1668. (14) Callahan, J. F.; Newlander, K. A.; Bugess, J. L.; Eggleston, D. S . ; Nichols, A.; Wong, A,; Huffman, W. F. Tetrahedron 1993, 49, 3479-3488. (15) Nagai, U.; Sato, K.; Nakamura, R.; Kato, R. Tetrahedron 1993,49, 3577-3592 and references cited therein. (16) Ripka, W. C.; De Lucca, G. V.; Bach, A. C., 11; Pottorf, R. S.; Blaney, J. M. Tetrahedron 1993, 49, 3593-3608. (17) Spatola, A. F.; Darlack, K. Tetrahedron 1988, 44, 821-833. (18) Morgan, B. A,; Gainor, J. A. In Annual Reports in Medicinal Chemistry; Vinick, F. J., Ed.; Pfizer, Inc., Central Research: Groton, CT, 1989; pp 243-252. f
bonding network, the electronic distribution, and the sterical hindrances, and therefore affect the conformational preferences. However, in comparison with peptides, the conformational studies (19) Davies, S. J. In Amino Acids and Peptides; Jones, S . H . , Ed.; The Royal Society of Chemistry: Cambridge, U.K., 1991; Vol. 22, pp 145-199. (20) Mock, W. L.; Zhang, J. Z. J. Org. Chem. 1990, 55, 5791-5793. (21) PaganiZecchini,G.;Paglialunga, M.;Torrini, I.; Lucente, G.;Gavuzzo, E.; Mazza, F.; Pochetti, G. Tetrahedron Lett. 1991, 32, 6779-6782. (22) Toniolo, C.; Bardi, R.; Piazzesi, A. M.; Jensen, 0. E.; Andersen, T. P.; Omar, R. S.;Senning, A,; La Cour, T. F. M. In Second Forum on Peptides, Colloque INSERM; Aubry, A.; Marraud, M., Vitoux, B., Eds.; John Libbey Eurotext: London, 1989; Vol. 174, pp 371-374. (23) Calgagni, A.; Gavuzzo,E.; Lucente, G.; Mazza, F.;Pochetti, G.; Rossi, D. Int. J. Pept. Protein Res. 1989, 34, 319-324. (24) Michel, G. A.; Ameziane-Hassani, C.; Boulay, G. Can.J. Chem. 1989, 67, 1312-1318. (25) Doi, M.; Takehara, S.;Ishida, T.; Inoue, M. Int. J. Pept. Protein Res. 1989, 34, 369-373. (26) Hollosi, M.; Zewdu, M.; Kollat, E.; Majer, Z.; Kajtar, M.; Batta, G.; Kover. K.: Sandor. P. Int. J. PeDt. Protein Res. 1990, 36, 173-181. (27) Sherman, D. B.; Spatola, A. F. J. Am. Chem. SOC.1990,112,433441. (28) Anwer, M. K.; Sherman, D. B.; Spatola, A. F. Int. J. Pept. Protein Res. 1990, 36, 392-399. (29) Dauber-Osguthorpe, P.; Campbell, M. M.; Osguthorpe, D. J. Int. J . Pept. Protein Res. 1991, 38, 357-377. (30) Calcagni, A,;Gavuzzo, E.; Lucente, G.; Mazza, F.; Pinnen, F.; Pochetti, G.; Rossi, D.Int. J. Pept. Protein Res. 1989, 34, 411-479. (31) Spatola, A. F.; Anwer, M. K.; Rockwell, A. L.; Gierasch, L. M. J. Am. Chem. SOC.1986, 108, 825-831. (32) Ma, S.; Richardson, J. F.; Spatola, A. F. J. Am. Chem. SOC.1991, 113, 8529-8530. (33) Ma, S.; Spatola, A. F. In Peptides: Chemistry and Biology; Smith, J. A., Rivier, J. E., Eds.; ESCOM: Leiden, The Netherlands, 1991; pp 777778. (34) Richman, S.; Goodman, M.; Nguyen, T.; Schiller, P. Int. J . Pept. Protein Res. 1985, 25, 648-662. (35) Hassan, M.; Goodman, M. Biochemistry 1986, 25, 7596-7606. (36) Di Bello, C.; Scatturin, A,; Vertuani, G.; D’Auria, G.; Gargiulo, M.; Paolillo, M.; Trivellone, E.; Gozzini, L.; De Castiglione, R. Biopolymers 1991, 31, 1397-1408. (37) Aumelas, A.; Rodriguez, M.; Heitz, A.; Castro, B.; Martinez, J. Int. J. Pept. Protein Res. 1991, 30, 596-604. (38) Van der Elst, P.; Elseviers, M.; De Cock, E.; Van Marsenille, M.; Tourw6, D.; Van Binst, G. Int. J. Pept. Protein Res. 1986, 27, 633-642. (39) Kasmierski, C. M.; Ferguson, R. D.; Knapp, R. J.; Lui, G. K.; Yamamura, H. I.; Hruby, V. J. Int. J. Pept. Protein Res. 1992,39,401-414. (40) Ma, S.; Spatola, A. F. Int. J. Pept. Protein Res. 1993, 41, 204-206. (41) Liang, G. B.; Dado, G. P.; Gellman, S . H. J. Am. Chem. SOC.1991, 113, 3994-3995. (42) Calcagni, A,;Gavuzzo, E.; Lucente, G.; Mazza, F.; Pinnen, F.; Pochetti, G.; Ross, D. Int. J. Pept. Protein Res. 1991, 37, 167-173.
0002-7863/93/ 1515-8898$04.00/0 0 1993 American Chemical Society
Conformational Perturbations of Peptides
J . Am. Chem. SOC.,Vol. 115, No. 20, 1993 8899
Table I. Free (Roman) and Bonded (Bold Italics) C=O, 0-H, and N-H Stretching Frequencies (cm-l)a for the N-Amino and N-Hydrooxy Analogues of Piv-Pro-NHMe
c=o compd P1 (Piv-Pro-NHMe) b C
Piv
Pro
1623w 160@ 1617s
1682s
BOC
b C
d A* 1 (Piv-Pro+[ CO-Nu(N@HMe)]NHMe)
b C
d
A'1 (Piv-Pro+[CO-N'(NPHBoc)]NHMe) b C
d
H 1 (Piv-Pro+[CO-N'(OflH)]NHMe) b C
d
H'1 (Piv-Pro+[CO-Nu(O~Me)]NHMe) b C
d
N@/OP-H
346OW
33343 1673s
3452M
159F d A1 (Piv-Pro+ [CO-N'(NBH2)] NHMe)
N-H
3328A
1618s
1 679s
1626s-e 1615S9e 1618s
1677s 1674s 1672s
336OW 3353w
1623s 1613s 1614s
1670s 1663s 168OS
33OOw 3309w
1625w 1619 161OS 161gS
168lS
1742s
3394w
328P 168OS 1677s
1589 1615w 158P 1616s
1658s
1624s 1613s 1615s
1672s 1667s 1665s
1655s 164P
1739s 1727s
3386s
3209 35OOw
3209
*
IR absorption: S,strong, M, medium; W, weak; B, broad. Solvent: CC14. Concentrations: 5 X l ( r M. Solvent: CHZC12. Concentrations: 5X M. Solvent: Me2SO. Concentrations: 5 X le3 M. Because of N-H solvation giving rise to a very broad ill-resolved band, the N-H stretching frequencies are not indicated in this solvent. e A medium absorption at 1605 cm-I disappearing on NP-methylation is due to the NPHz bending vibration.
of amide surrogate-containing peptide analogues are rather few in the literature.l7JW2 Contrary to N-methylation, N-amination and N-hydroxylation of the peptide bond have received little attention. As a matter of fact, natural peptides containing an N-amino or N-hydroxy substitution in the main chain are very Nevertheless, N-hydroxamide is known as a particularly strong proton donor capable of chelating metal cations in natural siderophores, and this property has been used for the design of potent enzyme inhibitors.46 The small number of N-amino and N-hydroxy analogues of bioactive peptides is due to several reasons; (i) the absence of N-hydroxylation or N-amination reagents compatible with peptides, (ii) the difficult obtention of optically pure a-hydrazino acids (NBHz-NaH-C*HR-COzH) and a-hydroxamino acids (OsH-NaH-C*HR-COzH), and (iii) the weak reactivity of their Na atom in most coupling reaction procedures.46947 After the pioneering work of Niedrich in the early 1970s on a-hydrazino acid-containing peptide analogues,47 new procedures for getting optically pure a-hydroxamino acids and a-hydrazino acids have been proposed,46,4*-52while the protecting groups and the coupling procedures have been diversified in peptide (43) Freidinger, R. M.; Lundell, G. F.; Bock, M. G.; Di Pardo, R. M.; Homnick, C. F.; Anderson,P. S.;Pettibone,D. J.;Clineschmidt, D. G.;Koupal, L.R.;Williamson, J. M.;Goetz,M. A.;Hensens,O. D.;Liesch, J. M.; Springer J. P. In Peptides: Chemistry, Structure and Biology; Rivier, J. E., Marshall, G. R., Eds.; ESCOM: Leiden, The Netherlands, 1990; pp 72-74. (44) McCullough, W. G.; Merkal, R. S. J . Bacteriol. 1976, 128, 15-20. (45) McCullough, W. G.; Merkal, R. S. J. Bacteriol. 1979,137,243-247. (46) Chimiak, A.; Milewska, J. Fortschr. Chem. Org. Narurst. 1988, 53, 203-277. (47) Niedrich, H.; Killer, G. J . Prakt. Chem. 1974, 316, 751-758 and references cited therein. (48) Genari, C.; Colombo, L.; Bertolini, G. J . Am. Chem. SOC.1986,108, 6394-6395. (49) Evans, D. A.; Britton, T. C.; Dorow, R. L.; Dellaria, J. F. J . Am. Chem. SOC.1986, 108,63956397. (50) Trimble, L. A.; Vederas, J. C. J . Am. Chem. SOC.1986, 108, 63976399. (51) Viret, J.; Gabard, J.; Collet, A. Tetrahedron 1987, 43, 891-894. (52) Vidal, J.; Drouin, J.; Collet, A. J . Chem. SOC.,Chem. Commun. 1991, 435437.
synthesis.s3 All these findings have encouraged us to consider the N-hydroxy and N-amino amide links as possible peptidomimetic groups.
In this paper, we report on the conformational properties of simple N-hydroxy and N-amino peptides deriving from the PivPro-NHR (Pl) and Piv-Pro-Gly-NHR (P2) peptides (Piv = Me3C-CO, R = Me or iPr), which are known to adopt preferentially the y- and 8-turn structures, respectively.54-5' The peptidomimetic group has been introduced either in the middle (in P2) or C-terminal (in P1 and P2) position. The Os-Me/Bzl and N@-Boc/Zsynthetic intermediates have also been examined. The pivalyl group has a 2-fold advantage over other acyl groups to shift the C-0 stretching to lower frequencies and to prevent cis-trans isomerization of the Pro-proceding amide b0nd.5495~All the derivatives have been investigated by lH-NMR and IR spectroscopy in organic solution rather than in water, which is known to solvate strongly such small molecules. Seven of them, having grown single crystals, have been examined in the solid state by X-ray diffraction. The experimental results have been complemented by a molecular dynamics simulation of the N-hydroxy analogues by using the SYBYL program.'* In the following, we use the IUPAC-recommended "+-bracket" nomenclature, in which the bracketed group is substituted for the amide bond in the cognate peptideS59 (53) Bodanski, M.; Bodanski, A. The Practice of Peptide Synthesis; Springer-Verlag: Berlin, 1984. (54) Boussard, G.; Marraud, M.; Aubry, A. Biopolymers 1979,18,12971331. (55) Aubry, A.; Protas, J.; Boussard, G.; Marraud, M. Acta Crystallogr., Sect. B 1980, 36, 2822-2824. (56) Boussard, G.; Marraud M.J . Am. Chem. SOC.1985,107,1825-1828. (57) Liang, G. B.; Rito, C. I.; Gellman, S. H. Biopolymers 1992,32,293301. (58) Clark,M.;Cramer,R. D., III;VanOpdenbosch,N.J. Comput. Chem. 1989, 10, 982-1012. (59) IUPAC-IUB Nomenclature Recommendations. Eur. J. Biochem. 1984, 138, 9.
Dupont et al.
8900 J. Am. Chem. SOC.,Vol. 115, No. 20, 1993
Table 11. Free (Roman) and Bonded (Bold Italics) C 4 , 0-H, and N-H Stretching Frequencies (cm-l)o for the N-Amino and N-Hydroxy Analogues of Piv-Pro-Gly-NHiPr C
"pd P2 (Piv-Pro-Gly-NHiPr) b C
4
N-H
Z
Piv
Pro
Gly
GlY
iPr
16121",160$ 1618w
1694s 1686s
1664s 1663s
1668s 1669s
1668s 1669s
1688s 1687s
1667s 1664s
1668s
1668s
1687M
1663s
l6w 1615M 1609
16705 1668s
1670s 1668s
1624?v' d 1619s*d
1678s 1680s
1665s 1668s
34w
3353w
1674s 1682s
165gS 1656s
3404
3317w
1654s
3435w 340lM
35OOw 32203
1623s
1691M 1679 1680M
1624M
1680M
1664s
1619M
1685M
1668s
3443s
N@/W-H
33403
I609 A2 (Piv-Pro$[CO-Na(NBH~)]Gly-NHiPr)
b C
16og.e 1616M 1 W
A'2 (Piv-Pro$[CO-Na(N@HZ)]Gly-NHiPr) 159p b 1616w C I@ H2 (Piv-Pro$ [CO-Na(OBH)] Gly-NHiPr) 1615w b
175lS 1734s
159P,158F C
1618M
3311s
3
w
33403
3369
3420M 33721",33W
35OOw 3319,3209
IMP H'2 (Piv-Pro$ [CO-Na(@Bzl)] Gly-NHiPr) b C
A3 (Piv-Pro-Gly$[ C0-Na(NBH2)]NHMe) b C
A*3 (Piv-Pro-Gly$[CO-Na(NBHMe)]NHMe) 1624s b 1619s C H3 (Piv-Pro-Gly$[CO-Na(OO-I)]NHMe) 1623M b
159P C
H'3 (Piv-Pro-Gly+ [C0-Na(08Me)] NHMe) b C
33323
1662s 344lW 34103
IR absorption: strong, S; medium, M; weak, W. Concentrations: 5 X lo-) M. Solvent: CHzCl2. Solvent: Me2SO. Because of N-H solvation giving rise to a very broad ill-resolved band, the N-H and 0-H stretching frequencies are not indicated in this solvent. A medium absorption at 1605 cm-1 disappearing on NB-methylation is due to the NBH2 bending vibration. e This absorption is only slightly reduced on NB-methylation and masks the NBH2 bending vibration. a
Experimental Section Synthesis. The compounds we have investigated are listed in Tables
I and 11. Direct coupling of Piv-Pro-OH or Piv-Pro-Gly-OH to OHNHMe using DCCI and DMAP as coupling reagents gave the H1 and H3 N-hydroxypeptides containing a C-terminal N-methylhydroxamide group. The homologous OB-methylated compounds H'1 and H'3 were prepared from MeO-NHZ by the same procedure. Derivatives H2 and H'2 with the hydroxamide link in the central position were obtained in good yields (75%) according to Figure 1. Although a preference has been stated for N-monosubstituted hydrazines,a the regioselective acylation of NBHrN'HMe and N B H r NaH-CH+20-OEt, HC1 (Aldrich) proved to depend on both the coupling partner and the coupling agent.61 For example, A3 was simply obtained from Piv-Pro-Gly-OSu and N&&NUHMe without protection. The same procedure failed for A1 and A2, probably because of the bulkier side chain of proline compared with glycine. The intermediate selective protection of the NB nitrogen was thus necessary for the synthesis of A1 (Figure 2), as already described for A2.6L All derivatives were purified by flash chromatography on silica gel, with CH~C12/isopropylalcohol as the eluent, and characterized by IH-NMR. 1H-NMR and IR Spectroscopy. The possible intramolecular hydrogen bonds were investigated by considering both the solvent sensitivity of the proton NMR signals in DMSO-&/C2HC13 mixtures and the stretching frequencies of the proton-donating and -accepting groups. We have focused our attention on the 0-H and N-H stretching frequencies (3 1003650cm-I) and the (tBu)C=O absorption in the 1580-1630~m-~domain, which is devoid of any other significant contribution (Tables I and 11). The peptide concentration in various solvents (CC4 for P1 analogues, CHzClz and DMSO for all derivatives) was adjusted to 5 X 10-4 M (60) Hegarty, A. F. In The Chemisfry of Hydraro, Azo, Azoxy Groups; Patai, S., Ed.; John Wiley and Sons: New York, 1975; pp 643-723. (61) Lecoq,A.;Marraud, M.;Aubry,A. Tefrahedronkrt.1991,32,27652768.
0
0
H
Figure 1. Synthesis of H2: a, iPrNH2/NMM/CH2C12, -15 ' C ; 6, BzlONH2, HCI/NMM/DMF, 25 OC; c, Piv-Pro-OH/DCCI/DMAP/ THF, 0 O C ; d, H2/5% Pd-C/MeOH.
(cc4)and 5 X lo-) M (CHZC12 and DMSO), for which further dilution confirmed the absence of molecular aggregation. IH-NMRspectra were run on a Briiker AC-2OOP apparatus. Chemical shifts were measured with reference to internal Me& and spin systems were solved by COSY experiments. Contrary to isolated hydroxamides,62 the N-hydroxy peptides only accommodate all-trans conformations. The (62) Kolasa, T. Terrahedron 1983, 39, 1753-1759.
J. Am. Chem. SOC.,Vol. 115, No. 20, 1993 8901
Conformational Perturbations of Peptides Table IU. Crystallographic Data
Piv-Pro+[C0-NOH] NHMe ( H l )
-
space group
Piv-Pro$[CO-NOH]Gly-NHiPr (H2) m12121 4 9.553(1) 10.890(2) 16.859(3)
E 1
z
Piv-Pro$[C0-N(NHZ)] Gly-NHiPr (A'2)
-
Pi
E 1 4 12.162(2) 9.938(2) 12.232(3)
p2 1 2 5.976(2) 9.362( 1) 16.154(1)
P21/c 4 15.525(2) 8.538(1) 12.254(2)
p2 I 2 9.730(1) 11,108(1) 11.227(1)
103.16(2)
91.59(2)
111.06(1)
91.56(1)
1.1 1 2708
1.15 1761
1.24 2724
1.22 2423
1819b
2174c
218ff
1664"
2312c
2279c
0.052
0.046 0.057 l/(u2(F) + 0.0077P)
0.065 0.083 1.311/(u2(F) + 0.0021P)
0.046 0.057 3.40/(u2(F) + 0.0005P)
0.057 0.064 l/(a2(F) + 0.076P)
0.054 0.070 1/(u2(F) 0.003P)
0.042 0.058 2.55/(a2(F) 0.0036P)
0.12 -0.19
0.26 -0.40
0.16 -0.16
0.20 -0.28
0.56 -0.33
0.16 -0.23
0.058
1.927/(u2(F) 0.0016P)
residual peak height max, e A-3 min, e A-3
-
1893"
105.68(1)
Y, deg
W
Piv-Pro$Piv-Pro-Gly$[CO-N(NH2)I - [CO-N(NHz)] Gly-NHiPr (A2) NHMe (A3)
1.19 1915
8, deg
d(calcd), g cm-3 no. of independent reflections no. of unique reflections final R final R w
-
1.15 2631
b, A
c, A a,deg
Piv-Pro+[CO-N(NHMe)] NHMe (A.1)
2 7.267(2) 9.775(2) 12.086(2) 72.54(2) 86.50(2) 68.71 (2) 1.24 2879
4 8.801(1) 10.147(2) 15.334(2)
a, A
Piv-Pro-Gly$[C0-NOH]NHMe (H3)
+
0.22 -0.19
+
+
" I > l.Su(1). b I > a(1). c z > 341).
/OH
N---( Fmoc'
H
'(
+
C 0
/ k i
+
H
N: H'
,
Me
,
,OH
C 0
A1
tBu\ C 0
Me
H
0
Figure 2. Synthesis of Al: a , ZCl/NMM/acetone, 0 'C; b, HCI 3N/ AcOEt and fractional crystallization; c, Boc20/DMAP/NMM/THF, 0 'C; d, H2/5% Pd-C/MeOH; e, SOC12/CH2C12 (acid chloride proce; dure5)); e', DCCI/CHzCI2 (symmetric anhydride p r o c e d ~ r e ~ ~ )Et2NH/CH2C12; g , tBuCOCl/NaOH; f ', H2/5% Pd-C/MeOH; g', tBuCOCI/NaOH; h, HCl 3N/AcOEt; i, NMM. same holds partly true for the N-amino peptides, since small amounts (never exceeding 30%) of cis conformers in chloroform-rich C2HCla/ DMSO-& mixtures are observed for A3 having a C-terminal N-amino group. IR spectra were scanned on a Brtiker IFS-85 apparatus in the Fourier transform mode using a cell path length of 2 mm (CCd), 0.5 mm (cc14 and CHzClz), or 0.2 mm (DMSO). The IR frequencies in low polar solvents (Tables I and 11) were assigned on the basis of previous results on similar compound^^^.^^.^^ and from the following considerations: (i) Free peptide N-Hs give a sharp absorption at 3400-3460 cm-', and bonded N-Hs give a stronger and broader contribution at 3300-3380 cm-'. (ii) FreepeptideC4vibrators havea strong andsharpabsorption at 1660--1690cm-' (butpivalamideC4stretchingsareshiftedto 15801630 cm-I), with a frequency shift of 10-30 cm-I in the bonded state.
(iii) Hydroxamides in the trans (E) conformation exhibit a free 0-H stretching frequency at 3500-3550 cm-l, and the C=O stretching is lowered by about 15 cm-' with reference to amides.62 (We have verified the absence of any additional absorption in the pivalamide C = O domain). (iv) N-amino amides have a very weak N-H absorption at 3330 cm-l and a C=O stretching frequency similar to that of classical amides. (v) In N-amino amides, the very weak contribution at 1600 cm-', which is eliminated by methylation of the N-amino group, is attributed to the NaH2 bending mode. In a strong solvating medium such as DMSO, the nonbonded, solvated N-H and 0-H absorptionsare considerablyenhanced and shifted to lower frequencies to such an extent that they mask the intramolecularly bonded contributions whereas the stretching frequencies of the bonded and nonbonded carbonyls are only little affected. X-ray Diffraction. Seven of the N-hydroxy and N-amino peptides havegrown singlecrystals from AcOEt/iPrzO solutions (Table 111). X-ray data were collected at room temperature on an Enraf Nonius CAD 4 automatic diffractometer with a graphite monochromator, operating with the Cu K a radiation (A = 1.540 5 1 A) in the 8-28 scanning mode (8 5 70'). Intensity data were corrected for Lorentz and polarization effects and for decay when three standard reflexionsdecreased by more than S I . Due to the small size (
