Structural comparison of homologous reduced peptide, reduced azapeptide, iminozapeptide, and methyleneoxypeptide analogues

Article abstract of DOI:10.1021/ja980937o

The homologous RCO-Pro-Xaa-NHR' model pseudodipeptides containing the reduced peptide (C(α)-CH₂NCH(α)), reduced azapeptide (C(α)CH₂NHN(α), methyleneoxy (C(α)CH₂OC(α)), and iminoazapeptide (C(α)CH=NN(α)) surrogate of the middle amide group have been prepared. Their structural analysis has been carried out in solution by ¹H NMR and IR spectroscopy and in the solid state by X-ray diffraction. The last three fragments, not protonated in the pH range 2-12, and the reduced fragment in its neural amine form induce quite similar molecular structures characterized by a hydrogen bond between NHR' and the N/O atom replacing the amide NH group and closing a five-membered cycle. The neutral amine or protonated ammonium state of the reduced amide fragment, with a pK(a) value of about 7, depends on the environment. Protonation induces a conformational transition due to the strong proton donating properties of the ammonium group which interacts with the RCO carbonyl.

Full text of DOI:10.1021/ja980937o

9444
J. Am. Chem. Soc. 1998, 120, 9444-9451
Structural Comparison of Homologous Reduced Peptide, Reduced
Azapeptide, Iminoazapeptide, and Methyleneoxypeptide Analogues
R. Vanderesse,*,† V. Grand,†, D. Limal,†,| A. Vicherat,† M. Marraud,† C. Didierjean,‡ and
A. Aubry‡
Contribution from the Laboratory of Macromolecular Physical Chemistry, UMR 7568 CNRS-INPL,
ENSIC-INPL, BP 451, 54001 Nancy, France, and Laboratory of Crystallography and Modeling of Mineral
and Biological Materials, ESA-7036, UniVersity Henri Poincare´ of Nancy, BP 236,
54509 VandoeuVre, France
ReceiVed March 19, 1998
Abstract: The homologous RCO-Pro-Xaa-NHR′ model pseudodipeptides containing the reduced peptide (CR-
CH2NHCR), reduced azapeptide (CRCH2NHNR), methyleneoxy (CRCH2OCR), and iminoazapeptide (CRCHd
NNR) surrogate of the middle amide group have been prepared. Their structural analysis has been carried out
in solution by 1H NMR and IR spectroscopy and in the solid state by X-ray diffraction. The last three fragments,
not protonated in the pH range 2-12, and the reduced fragment in its neutral amine form induce quite similar
molecular structures characterized by a hydrogen bond between NHR′ and the N/O atom replacing the amide
NH group and closing a five-membered cycle. The neutral amine or protonated ammonium state of the reduced
amide fragment, with a pKa value of about 7, depends on the environment. Protonation induces a conformational
transition due to the strong proton donating properties of the ammonium group which interacts with the RCO
carbonyl.
Various amide surrogates have been used for the design of
number of experimental4,5 and theoretical6 conformational
analyses. The crystal structures of the NR-Z and NR-Boc-
Proψ[CH2NH]Leu-Gly-NH2 reduced derivatives of the oxytocin
C-terminal tripeptide,7 containing a reduced Pro-Leu amide
bond, have been solved,8 as well as the structures of reduced
protease inhibitors in renin2b,d or endothiapepsin9 complexes.
Surprisingly, most of these studies do not take into account the
possibility for the reduced amide bond to be protonated at the
physiological pH. This point is of importance since experi-
mental results have demonstrated that protonation of a reduced
amide link affects considerably its conformational properties.5e,10
peptidase inhibitors.1 The reduced amide bond CH2-NH,
considered as mimicking the C(OH)2-NH transient state of the
amide bond CO-NH during enzymatic cleavage, is frequently
used,2 probably because of its easy introduction in a peptide
chain by reductive amination of a peptide aldehyde.3 However,
there is some NMR indication that the pKa of a reduced amide
is at about neutral pH,4 and the question arises about the neutral
or ionic state of a reduced peptide at the physiological pH.
The reduced peptides have been the subject of a limited
† ENSIC-INPL.
‡ University Henri Poincare´ of Nancy.
(5) (a) Di Bello, C.; Scatturin, A.; Vertuani, G.; D′Auria, G.; Gargiulo,
M.; Paolillo, L.; Trivellone, G.; Gozzoni, L.; De Castiglione, R. Biopolymers
1991, 31, 1397-1408. (b) Kazmierski, W. 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. (c) Vander Elst, P.; Elseviers, M.; De Cock, E.; Van
Marsenille, M.; Tourwe´, D.; Van Binst, G. Int. J. Pept. Protein Res. 1986,
27, 633-642. (d) Ma, S.; Spatola, A. F. In Peptides: Chemistry and Biology;
Smith, J. A., Rivier, J. E., Eds.; ESCOM: Leiden, The Netherlands, 1992;
pp 777-778. (e) Ma, S.; Spatola, A. F. Int. J. Pept. Protein Res. 1993, 41,
204-206.
Present address: Laboratory for Scientific Police, BP 2162, 31021
Toulouse Cedex 2, France.
| Present address: IBMC, 67084 Strasbourg Cedex, France.
¶ Abbreviations: Boc, tert-butyloxycarbonyl; DIEA, diisopropylethy-
lamine; ICF, isobutyl chloroformate; NMM, N-methylmorpholine; PE,
petroleum ether; Piv, pivaloyl; TFA, trifluoroacetic acid; Z, benzyloxycar-
bonyl.
(1) Gante, J. Angew. Chem., Int. Ed. Engl. 1994, 33, 1699-1720 and
references therein.
(2) (a) Szelke, M.; Leckie, B. J.; Tree, M.; Brown, A.; Grant, J.; Hallett,
A.; Hughes, M.; Jones, D. M.; Lever, A. F. Hypertension, Suppl. II 1982,
4, 52-69. (b) Epps, D. E.; Mao, B.; Staples, D. J.; Sawyer, T. K. Int. J.
Pept. Protein Res. 1988, 31, 22-34. (c) Sawyer, T. K.; Pals, D. T.; Mao,
B.; Maggiora, L. L.; Staples, D. J.; De Vaux, A. E.; Shostarez, H. J.; Kinner,
J. H.; Smith, C. W. Tetrahedron 1988, 44, 661-673. (d) Sawyer, T. K.;
Pals, D. T.; Mao, B.; Staples, D. J.; De Vaux, A. E.; Maggiora, L. L.;
Affholter, J. A.; Kati, W.; Duchamp, D.; Hester, J. B.; Smith, C. W.; Saneii,
H. H.; Kinner, J.; Handschumacher, M.; Carlson, W. J. Med. Chem. 1988,
31, 18-30. (e) Kaltenbronn, J. S.; Hudspeth, J. P.; Lunney, E. A.;
Michniewicz, B. M.; Nicolaides, E. D.; Repine, J. T.; Roark, W. H.; Stier,
M. A.; Tinner, F. J.; Woo, P. K. W.; Essenburg, A. D. J. Med. Chem. 1990,
33, 838-845.
(3) (a) Szelke, M.; Leckie, B.; Hallet, A.; Jones, D. M.; Sueiras, J.;
Atrasch, B.; Lever, A. F. Nature 1982, 299, 555-557. (b) Martinez, J.;
Bali, J. P.; Rodriguez, M.; Castro, B.; Magous, R.; Laur, J.; Lignon, M. F.
J. Med. Chem. 1985, 28, 1874-1879.
(4) Aumelas, A.; Rodriguez, M.; Heitz, A.; Castro, B.; Martinez, J. Int.
J. Pept. Protein Res. 1987, 30, 596-604.
(6) (a) Dauber-Osguthorpe, P.; Jones, D. K.; Campbell, M. M.; Os-
guthorpe, D. J. Tetrahedron Lett. 1990, 31, 917-920. (b) Dauber-
Osguthorpe, P.; Campbell, M. M.; Osguthorpe, D. J. Int. J. Pept. Protein
Res. 1991, 38, 357-377.
(7) In the following, we use Spatola’s nomenclature for pseudopeptides
where the bracketed group is substituted for the amide CO-NH group
(Spatola, A. F. In Chemistry and Biochemistry of Amino Acids, Peptides
and Proteins; Weinstein, B., Ed.; Marcel Dekker Inc.: New York, 1983;
Vol. 7, pp 267-357). In addition, an amino acid analogue is noted by putting
in quotes the corresponding three-letter code, and the methylene carbon in
a reduced amino acid analogue is noted Cr. The AzAla and AzGly codes
specify the aza analogues of alanine and glycine, respectively.
(8) Toniolo, C.; Valle, G.; Crisma, M.; Kaltenbronn, J. S.; Repine, J.
T.; Van Binst, G.; Elseviers, M.; Tourwe´, D. Pept. Res. 1989, 2, 332-337.
(9) Cooper, J.; Foundling, S.; Hemming, A.; Blundell, T.; Jones, D. M.;
Hallett, A.; Szelke, M. Eur. J. Biochem. 1987, 169, 215-221.
(10) (a) Grand, V.; Aubry, A.; Dupont, V.; Vicherat, A.; Marraud, M. J.
Pept. Sci. 1996, 2, 381-391. (b) Geyer, A.; Mu¨ller, G.; Kessler, H. J. Am.
Chem. Soc. 1994, 116, 7735-7743.
S0002-7863(98)00937-8 CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/09/1998

Amide Surrogates
J. Am. Chem. Soc., Vol. 120, No. 37, 1998 9445
Table 1. List of the Pseudopeptides Investigated with the
Derivatives Reproducing the N- or C-Terminal Part of the
Moleculesa
Chart 1. General Formula of the Investigated Reduced
Dipeptide (X-Y-ZR ) CrH2NHCRHMe),
Methyleneoxypeptide (X-Y-ZR ) CrH2OCRH2),
Iminoazapeptide (X-Y-ZR ) CrH)NNRMe), and Reduced
Azapeptide (X-Y-ZR ) CrH2NHNRMe) Analogues.
compd
code
techniques
ref
Neutral Reduced Peptides
Piv-Proψ[CH2NH]Ala-NHiPr
Piv-Proψ[CH2NZ]Ala-NHiPr
Piv-Proψ[CH2NH]NHiPr
nBu2NCH2CONHMe
1
1′
1N
1C
IR, 1H NMR, X-ray
X-ray
IR
b
b
10a
15
IR
Protonated Reduced Peptidesc
Piv-Proψ[CH2N+H2]Ala-NHiPr 1+
IR, 1H NMR
b
10a
15
Piv-Proψ[CH2N+H2]NHiPr
nBu2N+HCH2CONHMe
Methylenoxypeptide
1+N IR
1+C IR
A neutral amine is essentially a proton-accepting site by its
nitrogen lone pair and a weak donating site, whereas an
ammonium is exclusively a strong proton donor. In particular,
the ammonium group is capable of interacting with the peptide
carbonyls and favoring â- and γ-like turns in the chain.10a
The phosphonamide, phosphinic, sulfinamide, and sulfona-
mide groups11 have been also proposed as surrogates of the
amide tetragonal transient state, but their difficult synthesis is
an obstacle to a general use. A noncleavable, easy to obtain,
nonprotonable peptide surrogate would be of interest to the
design of bioresistant peptide analogues. The lower pKa value
of 3.53 for protonated semicarbazide12 indicates that it is not
protonated at the physiological pH. We therefore have exam-
ined the structural properties of the RCO-Pro-Xaa-NHR′ dipep-
tide analogues containing a modified Pro-Xaa link and having
the general formula in shown Chart 1, where RCO ) Boc or
Piv-Proψ[CH2O]Gly-NHiPr
2
2C
IR, 1H NMR
IR, 1H NMR
b
b
MeOCH2CONHiPr
Iminoazapaptides
Piv-Proψ[CHdN]AzAla-NHiPr
Boc-Proψ[CHdN]AzGly-NH2
iPrCHdNNMeCONHiPr
3
3′
3C
IR, 1H NMR, X-ray
X-ray
b
b
b
IR, 1H NMR
Reduced Azapeptide
Piv-Proψ[CH2NH]AzAla-NHiPr
iBuNHNMeCONHiPr
4
4C
IR, 1H NMR, X-ray
b
b
IR, 1H NMR
a For nomenclature, see ref 7. b This work. c PF6 is the associated
-
anion.
i
Piv, R′ ) H or Pr, X-Y-ZR ) CrH2NHCR (neutral reduced
peptide), CrH2N+H2CR (protonated reduced peptide), CrH2NHNR
(reduced azapeptide), CrH2OCR (methyleneoxypeptide), or CrHd
NNR (iminoazapeptide), where Cr denotes the “reduced” carbon
replacing the peptide carbonyl. The structural analysis has been
carried out on the pseudopeptides listed in Table 1 and on the
compounds reproducing the N- and C-terminal part of the
molecules by using 1H NMR, IR spectroscopy, and X-ray
diffraction. Such small molecules are readily solvated by water,
where they do not exhibit defined spectroscopic data. We
therefore have considered organic solvents (CH2Cl2, CHCl3,
MeCN, and DMSO) with increasing polarity for the spectro-
scopic experiments.
Experimental Procedures
Synthesis. The reduced dipeptide 1 was obtained by the reductive
amination procedure3b by coupling the aldehyde 513 to the L-alanine
methyl ester (Figure 1). To make the chromatographic purification
easier and to prevent undesirable side reactions during further acylation
steps, the reduced amide was Z-protected to give 6. The ester function
in 6 was saponified and converted into the isopropylamide group in 7
by the mixed anhydride method. The Piv group was substituted for
Boc in 1′, and the Z group was eliminated by catalytic hydrogenolysis
to yield the neutral reduced dipeptide 1, which was then protonated
Figure 1. Synthesis of the neutral 1 and protonated 1+ reduced
dipeptide by reductive amination and intermediate Z protection (1′) of
the reduced amide bond. a: (1) HCl‚H-Ala-OMe, (2) NaBH3CN, (3)
i
ZCl/NMM; b: (1) 1 N NaOH/acetone, (2) ICF/NMM, (3) PrNH2; c:
(1) HCl/AcOEt, (2) Piv-Cl/NMM; d: H2/5% Pd-C; e: Et2O+H.PF6
.
-
quantitatively to 1+ by PF6-‚Et2O+H (Aldrich 17,638-9). The PF6
-
anion was selected to obtain an ammonium salt soluble in nonpolar
media and for its weak interaction with ammonium anions,14 thus
allowing the ammonium group to participate in intramolecular interac-
tions.15 The methyleneoxypeptide 2 was prepared from the com-
mercially available Boc-prolinol 10 (Merck 818348), which was treated
by NaH and reacted with N-isopropyl-bromoacetamide (9) (Figure 2).16
The synthesis of the iminoazadipeptides 3 and 3′ and the reduced
azadipeptide 4 is summarized in Figure 3. Coupling of Boc-prolinal
516 either to semicarbazide 11 or to the substituted semicarbazide 13
gave the iminoazadipeptides 3′ and 14, respectively. Substitution of
the Piv group for Boc in 3, followed by catalytic reduction of the imine
(11) (a) Elliott, R. L.; Marks, N.; Berg, M. J.; Portoghese, P. S.
Biochemistry 1985, 28, 1208-1216. (b) Bartlett, P. A.; Marlowe, C. K.
Biochemistry 1987, 26, 8553-8561. (c) Parson, W. H.; Patchett, A. A.;
Bull, H. G.; Schoen, W. R.; Taub, D.; Davidson, J.; Combs, P. L.; Springer,
J. P.; Gadebusch, H.; Weissberger, B.; Valiant, M. E.; Mellin, T. N.; Busch,
R. D. J. Med. Chem. 1988, 31, 1772-1778. (d) Morgan, B. P.; Scholtz, J.
M.; Ballinger, M. D.; Zipkin, I. D.; Bartlett, P. A. J. Am. Chem. Soc. 1991,
113, 297-307. (e) Phillips, M. A.; Kaplan, A. P.; Rutter, W. J.; Bartlett, P.
A. Biochemistry 1992, 31, 959-963. (f) Moree, W. J.; Van der Marel, G.
A.; Liskamp, R. M. J. Tetrahedron Lett. 1991, 32, 409-412. (g) Merricks,
D.; Sammes, P. G.; Walker, E. R. H.; Henrick, K.; McPartlin, M. J. Chem.
Soc., Perkin Trans. 1 1991, 2169-2176. (h) Calvagni, A.; Gavuzzo, E.;
Lucente, G.; Mazza, F.; Pinnen, F.; Pochetti, G.; Rossi, D. Int. J. Pept.
Protein Res. 1991, 37, 167-173.
(14) Moreno-Gonzalez, E.; Marraud, M.; Ne´el, J. J. Chim. Phys., Phys.-
Chim. Biol. 1977, 74, 563-571.
(15) Moreno-Gonzalez, E.; Marraud, M. J. Chim. Phys., Phys.-Chim.
Biol. 1980, 77, 149-155.
(12) Goddard, D. R.; Lodam, B. D.; Ajavi, S. O.; Campbell, M. J. J.
Chem. Soc. (A) 1969, 506-512.
(13) Fehrentz, J. A.; Castro, B. Synthesis 1983, 676-678.
(16) Breton, P.; Monsigny, M.; Meyer, R. Int. J. Pept. Protein Res. 1990,
35, 346-351.

9446 J. Am. Chem. Soc., Vol. 120, No. 37, 1998
Vanderesse et al.
Piv-Proψ[CrH2NZ]Ala-NHiPr. 1′ (Figure 1) was purified by silica
gel flash chromatography (AcOEt/PE 60/40) and recrystallized from
AcOEt/PE. White crystals: mp ) 118-120 °C, Rf ) 0.36 (iPrOH/
CH2Cl2 3/97) or 0.31 (AcOEt/PE 50/50), 20% overall yield. IR (CH2-
Cl2, cm-1): 3280 (iPr-NH); 1701 (Z-CO); 1675 (Ala-CO); 1611 (Piv-
CO). 1H NMR (DMSO-d6, ppm), the two conformers I (35%) and II
(65%) denote cis-trans isomerization of the Z-N amide bond: 0.86
(I) and 1.03 (II) (d, 3J ) 6.7 Hz, 6H, iPr-(CH3)2); 0.95 (I) and 1.14 (II)
3
(s, 9H, Piv-(CH3)3); 1.39 (d, J ) 7.2 Hz, Ala-CâH3); 1.54-2.09 (m,
2
4H, “Pro”-CâH2 + CγH2); 3.26 (B) and 3.41 (A) (ABX, JAB ) 14.1
3
3
Hz, JBX ) 7.8 Hz, JAX ) 5.7 Hz, 2H, “Pro”-CrH2); 3.58 (m, 2H,
“Pro”-CδH2); 3.79 (m, 1H, Pr-CH); 4.09 (q, J ) 7.2 Hz, 1H, Ala-
i
3
CRH); 4.33 (m, 1H, “Pro”-CRH); 4.85 (I) and 5.20 (II) (b, 2H, Z-CH2);
i
7.33 (b, 5H, Z-C6H5); 7.48 (I) and 7.89 (II) (b, 1H, Pr-NH).
Piv-Proψ[CrH2NH]Ala-NHiPr. 1 (Figure 1) was crystallized from
AcOEt/PE. White crystals: mp ) 82-84 °C, Rf ) 0.50 (iPrOH/CH2-
Cl2 10/90), 90% yield. IR (DMSO, cm-1): 3280 (iPrNH), 1613 (Piv-
CO), 1665 (Ala-CO). 1H NMR (CDCl3, ppm): 1.15 and 1.16 (d, 3J )
i
3
6.7 Hz, 6H, Pr-(CH3)2); 1.25 (s, 9H, Piv-(CH3)3); 1.27 (d, J ) 6.7
Hz, Ala-CâH3); 1.72 (m, 1H, “Pro”-CâH); 1.80-2.02 (m, 3H, “Pro”-
CâH + CγH2); 2.36 (b, 1H, Ala-NH); 2.36 (B) and 2.83 (A) (ABX,
2JAB ) 11.4 Hz, 3JBX ) 7.7 Hz, 3JAX ) 4.1 Hz, 2H, “Pro”-CrH2); 3.16
(q, 3J ) 6.7 Hz, 1H, Ala-CRH); 3.49-3.74 (m, 2H, “Pro”-CδH2); 4.05
(m, 1H, iPr-CH); 4.17 (m, 1H, “Pro”-CRH); 7.16 (d, 3J ) 7.5 Hz, 1H,
iPr-NH).
Piv-Proψ[CrH2N+H2]Ala-NHiPr.PF6-. 1+ (Figure 1) was recov-
ered as a white powder by lyophilization. IR (DMSO, cm-1): 3280
(iPr-NH), 1616 (Piv-CO), 1685 (Ala-CO). 1H NMR (CDCl3, ppm):
Figure 2. Synthesis of the methyleneoxy peptide 2.16 a: iPrNH2; b:
NaH/THF; c: (1) H2/5% Pd-C, (2) Piv-Cl/NMM.
i
1.19 and 1.22 (2d, 3J ) 6.6 Hz, 6H, Pr-(CH3)2); 1.29 (s, 9H, Piv-
3
(CH3)3); 1.52 (d, J ) 6.7 Hz, Ala-CâH3); 1.50 (m, 1H, “Pro”-CâH);
1.80-2.30 (m, 3H, “Pro”-CâH + CγH2); 3.11 (b, 2H, “Pro”-CrH2); 3.48
3
(q, J ) 6.7 Hz, 1H, Ala-CRH); 3.60 and 3.86 (2m, 2H, “Pro”-CδH2);
4.03 (m, 1H, iPr-CH); 4.42 (m, 1H, “Pro”-CRH); 7.27 (b, 1H, iPr-NH);
8.18 and 9.78 (2b, 2H, Ala-N+H2).
Piv-Proψ[CrH2O]Gly-NHiPr. 2 (Figure 2) was recovered as a
colorless oil after silica gel chromatography with AcOEt/PE (30/70):
Rf ) 0.35; 50% overall yield. IR (CH2Cl2, cm-1): 3414 (iPr-NH), 1614
(Piv-CO), 1673 (Ala-CO). 1H NMR (CDCl3, ppm): 1.20 (d, 3J ) 6.6
Hz, 6H, iPr-(CH3)2); 1.26 (s, 9H, Piv-(CH3)3); 1.40-2.00 (m, 4H, “Pro”-
CâH2 + CγH2); 3.40-3.80 (m, 4H, “Pro”-CδH2 + CrH2); 3.96 (m, 2H,
i
Gly-CRH2); 4.13 (m, 1H, Pr-CH); 4.37 (m, 1H, “Pro”-CRH); 6.64 (b,
i
1H, Pr-NH).
MeOCH2CONHiPr. 2C was obtained as a colorless oil from
MeOCH2CO2H (Aldrich 19,455-7) by the mixed anhydride method
i
using ICF, NMM, and PrNH2. It was purified by silica gel chroma-
tography using iPrOH/CH2Cl2 as eluent (Rf ) 0.40). IR (CH2Cl2, cm-1):
3
3411 (NH), 1674 (CO). 1H NMR (CDCl3, ppm): 1.19 (d, J ) 6.5
i
Hz, 6H, Pr-(CH3)2); 3.42 (s, 3H, OCH3); 3.87 (s, 2H, CH2); 4.14 (m,
i
i
1H, Pr-CH); 6.33 (b, 1H, Pr-NH).
Boc-Proψ[CHdN]AzGly-NH2 (3′) (Figure 3). A solution of Boc-
prolinal 5 (6.5 mmol), semicarbazide 11 (9.75 mmol), and AcONa (26
mmol) in EtOH (15 mL) was stirred overnight at room temperature in
the presence of molecular sieves. 3′ was purified by silica gel flash
chromatography with iPrOH/CH2Cl2 (5/95) as the eluent and crystallized
from EtOH. White solid: mp ) 161 °C, Rf ) 0.38 (iPrOH/CH2Cl2
5/95), 85% yield. IR (CH2Cl2, cm-1): 1567 (CdN), 1687 (AzGly-
CO); 1700 (Boc-CO); 3461, 3528, 3410, and 3355 (NH2 + NH). 1H
NMR (CDCl3, ppm), I (30%) and II (70%) are two conformers due to
cis-trans isomerization of the Boc-“Pro” amide link: 1.41 (I) and 1.46
(II) (s, 9H, Boc-(CH3)3); 1.60-2.20 (m, 4H, “Pro”-CâH2 + CγH2); 3.41
(m, 2H, “Pro”-CδH2); 4.30 (I) and 4.40 (II) (m, 1H, “Pro”-CRH); 5.50
(b, 2H, NH2); 7.00 (I) and 7.08 (II) (m, 1H, NdCH); 9.17 (I) and 9.51
(II) (b, 1H, N-NH).
Figure 3. Synthesis of the iminoazapeptides 3 and 3′ by condensation
of Boc-prolinal and a semicarbazide, and reduction of 3 into the reduced
azadipeptide 4: (a) (1) OdCdNiPr, (2) HCl/AcOEt; (b) AcO-Na+/
EtOH/molecular sieves/room temperature (rt); (c) NMM/AcO-Na+/
EtOH/molecular sieves/rt; (d) (1) TFA/CH2Cl2 40/60, (2) Piv-Cl/NMM;
(e) H2/5% Pd-C/EtOH/AcOH.
Piv-Proψ[CHdN]AzAla-NHiPr (3) (Figure 3). To methylhydra-
zine (12) (20 mmol) in Et2O (30 mL) was added OdCdNiPr (20 mmol)
in Et2O (10 mL) under stirring at -5 °C, and the solution was allowed
to come back to room temperature. 13 is precipitated by HCl-saturated
bond, gave the reduced azadipeptide 4. The Piv group in 1-4 was
introduced to exclude the cis conformation of the Pro-preceding amide
bond.17 All intermediate and final derivatives were purified by flash
(17) (a) Nishihara, H.; Nishihara, K.; Uefuji, T.; Sakota, N. Bull. Chem.
Soc. Jpn 1975, 48, 553-555. (b) Aubry, A.; Cung, M. T.; Marraud, M. J.
Am. Chem. Soc. 1985, 107, 7640-7647. (c) Liang, G. B.; Rito, C. J.;
Gellman, S. H. Biopolymers 1992, 32, 293-301.
1
chromatography and characterized by H NMR and IR spectroscopy.
The melting points were uncorrected. Rf values were measured by thin-
layer chromatography (Merck, Silicagel 60 F254 plates 5735).

Amide Surrogates
J. Am. Chem. Soc., Vol. 120, No. 37, 1998 9447
Table 2. Crystallographic Data
pseudopeptide
1a
1′ a
3a
3′ a
4b
P21/c
space group
Z
P212121
4
P212121
4
P1
1
P212121
12
4
cell dimensions
a (Å)
b (Å)
9.337(1)
9.458(1)
20.425(3)
11.095(1)
25.617(3)
8.787(1)
5.951(1)
9.387(1)
15.920(2)
92.98(1)
98.20(1)
105.58(1)
1.14
10.022(1)
16.485(2)
25.765(3)
10.083(2)
18.553(1)
10.282(1)
c (Å)
R (deg)
â (deg)
111.19(2)
γ (deg)
calcd density (g cm-3
no. of total reflns
)
1.10
1785
1.15
1.24
1.11
2722
3139
4374
3632
no. of significant reflns 1529c
residual factors
2265c
1744c
3136c
3357d
R
Rw
w
0.055
0.059
0.054
0.059
0.060
0.067
0.046
0.055
0.073e
1/[σ2(F) + 0.005F2] 2.953/[σ2(F) + 0.0007F2] 0.3774/[σ2(F) + 0.0180F2] 0.0462/[σ2(F) + 0.0023F2]
residual peak height
max (e Å-3
)
0.20
-0.25
0.17
-0.25
0.32
-0.25
0.24
-0.30
0.34
-0.36
min (e Å-3
)
a Refined by SHELX86.19 b Refined by SHELXL93.20 c I > 3 σ(I). d I > 2 σ(I). e wR ) 0.219.
AcOEt and crystallized from EtOH (white solid, mp ) 177 °C, 80%
yield). Treatment of 5 by 13 using the same procedure as for 3′ gave
14. The Boc group was cleaved by TFA, and the Piv group was
introduced by PivCl and DIEA to give 3, which was crystallized from
AcOEt. White solid: mp ) 70 °C. Rf ) 0.48 (iPrOH/CH2Cl2, 5/95),
85% yield. IR (CH2Cl2, cm-1): 1516 (CdN); 1618 (Piv-CO); 1672
CO stretching frequencies were assigned on the basis of previous studies
on related peptides.17b In particular, the free amide NH is expected to
give a sharp absorption in the 3400-3450 cm-1 domain, and the free
Piv-CO, a strong contribution at 1610-1625 cm-1 in CH2Cl2. Previous
studies on ammonia and protonated reduced peptides have shown that
the N+H2 stretching frequencies greatly depend on the solvent and the
i
n
(AzAla-CO); 3417 (NH). 1H NMR (CDCl3, ppm): 1.16 (d, 6H, Pr-
associated anion.10a,15 For example, the Bu2N+H2 group exhibits a
(CH3)2); 1.28 (s, 9H, Piv-(CH3)3); 1.90-2.10 (m, 4H, “Pro”-CâH2 +
CγH2); 3.14 (s, 3H, AzAla-CâH3); 3.70 (m, 2H, “Pro”-CδH2); 3.94 (m,
1H, iPr-CH); 4.86 (m, 1H, “Pro”-CRH); 6.23 (d, 3J ) 7.6 Hz, 1H, NH);
sharp peak at 3246 cm-1 when associated with the PF6- anion in a low
polar solvent (CH2Cl2) but gives rise to a multicomponent absorption
shifted down to 2700 cm-1 when solvated in a strong aprotic medium.
The nBu2N+H2 cation has also a weak absorption at 1596 cm-1 in CH2-
Cl2, while the PF6- anion has no visible absorption in the 2200-2800
and 3100-3600 cm-1 domains.
3
6.79 (d, J ) 4.0 Hz, 1H, NdCH).
iPrCHdNNMeCONHiPr. 3C was prepared from isobutyraldehyde
and 13 using the same procedure as for 3 to give a colorless oil by
silica gel flash chromatography with AcOEt/PE (25/75) as the eluent
(Rf ) 0.41). IR (CH2Cl2, cm-1): 1516 (CdN); 1669 (AzAla-CO); 3413
(NH). 1H NMR (CDCl3, ppm): 1.06 (d, 3J ) 6.9 Hz, 6H, NiPr-(CH3)2);
1H NMR spectra were run on a Bruker AC-200P apparatus with
Me4Si as internal reference, and the spin systems were solved by COSY
and HOHAHA experiments. The solvent accessibility, and therefore
the free or hydrogen-bonded character of the amide and ammonium
protons in these small molecules, was investigated by considering their
resonance shift in CDCl3/DMSO-d6 mixtures with increasing DMSO-
d6 content.17b The signal of a hydrogen-bonded, solvent-shielded NH
is only slightly sensitive to DMSO content, whereas the signal of a
free, solvent-exposed NH is downfield shifted from small DMSO-d6
contents.
X-ray Diffraction. Single crystals of the reduced dipeptides 1 and
1′, the iminoazadipeptides 3 and 3′, and the reduced azadipeptide 4
were grown by slow evaporation of an EtOH/AcOEt solution. The
X-ray diffraction data were collected on an Enraf Nonius CAD-4 four-
circle diffractometer in the ω/2θ scan mode. The independent
reflections were measured at room temperature in the 1-70° θ range
using Cu KR radiation (λ ) 1.541 78 Å) monochromatized by a graphite
crystal. During data collection, two standard reflections were measured
every 2 h to check the stability of the crystal. Intensities were corrected
for Lorentz and polarization effects, but no absorption correction was
applied. Crystallographic data are given in Table 2. The crystal
structures were solved by direct methods using SHELXS90.18 The E
maps revealed the whole molecules apart from the hydrogen atoms
(three independent molecules of 3′ are present in the cell), and the
structures were refined through the least-squares procedure with the
complete matrix of normal equations using SHELX8619 for 1, 1′, 3,
and 3′ and SHELXL9320 for 4. Heavy atoms were affected by
anisotropic thermal factors, and hydrogen atoms were located on E
map differences and affected by an isotropic thermal factor of 4 Å2.
The residual R factors are indicated in Table 2. The NH hydrogen
3
1.31 (d, J ) 6.8 Hz, 6H, CiPr-(CH3)2); 2.34 (m, 1H, CiPr-CH); 3.07
(s, 1H, N-CH3); 3.90 (m, 1H, iPr-CH); 6.27 (d, 3J ) 6.0 Hz, 1H, NH);
3
6.66 (d, J ) 5.1 Hz, 1H, dCH).
Piv-Proψ[CrH2NH]AzAla-NHiPr (4) (Figure 3). Catalytic hydro-
genation (96 h) on Pd-C 10% (10 mg) under atmospheric pressure of
3 (40 mg, 0.14 mmol) in MeOH (10 mL) and AcOH (0.2 mL) gave 4,
which was crystallized from AcOEt. White solid: mp ) 78 °C, Rf )
0.40 (iPrOH/CH2Cl2, 5/95), 90% yield. IR (CH2Cl2, cm-1): 1612 (Piv-
CO); 1653 (AzAla-CO); 3404 (N-H). 1H NMR (CDCl3, ppm): 1.16
i
(d, 3J ) 6.5 Hz, 6H, Pr-(CH3)2); 1.28 (s, 9H, Piv-(CH3)3); 1.80-2.10
(m, 4H, “Pro”-CâH2 + CγH2); 2.62 (B) and 3.12 (A) (ABX, JAB
)
11.3 Hz, JAX ) 4.5 Hz, JBX ) 7.0 Hz, 2H, “Pro”-CrH2); 3.04 (s, 3H,
AzAla-CâH3); 3.55 (m, 2H, “Pro”-CδH2); 3.64 (m, 1H, N-NH); 4.20
3
(m, 1H, “Pro”-CRH); 6.19 (d, J ) 7.7 Hz, 1H, NH).
iBuNHNMeCONHiPr. 4C was obtained from 3C by the same
procedure as for 4 from 3. Purification by silica gel chromatography
gave a colorless oil with AcOEt/PE (50/50) as the eluent (Rf ) 0.26).
IR (CH2Cl2, cm-1): 1669 (CO); 3413 (NH). 1H NMR (CDCl3, ppm):
3
i
3
i
0.94 (d, J ) 6.7 Hz, 6H, Pr-(CH3)2); 1.12 (d, J ) 6.6 Hz, 6H, Bu-
i
3
i
(CH3)2); 1.64 (m, 1H, Bu-CH); 2.58 (d, J ) 6.6 Hz, 2H, Bu-CH2);
3.02 (s, 3H, N-CH3); 3.85 (m, 1H, iPr-CH); 6.24 (d, 3J ) 7.2 Hz, 1H,
NH).
1H NMR and FTIR Spectroscopy. Spectroscopic experiments were
carried out on the neutral reduced peptide 1 and its ionic form 1+, the
methyleneoxypeptide 2, the iminoazapeptide 3, and the reduced
azapeptide 4, all containing the Piv group, which is known to favor
the trans isomer of the Piv-Pro amide bond.17 IR spectra were scanned
in the Fourier transform mode on a Bruker IFS-25 apparatus using a
cell path length of 0.5 mm to investigate the NH (3200-3500 cm-1),
CO (1580-1720 cm-1), and N + H (2500-3300 cm-1) stretching
frequencies. The peptide concentration was 0.005 M, and further
dilution confirmed the absence of molecular aggregation. The NH and
(18) Sheldrick, G. M. SHELX86. Program for the solution of crystal
structures; University of Go¨ttingen: Go¨ttingen, Germany, 1986.
(19) Sheldrick, G. M. Acta Crystallogr., Sect. A 1990, 46, 467-473.
(20) Sheldrick, G. M. SHELXL93. Programs for the refinement of crystal
structures; University of Go¨ttingen: Go¨ttingen, Germany, 1993.

9448 J. Am. Chem. Soc., Vol. 120, No. 37, 1998
Vanderesse et al.
Figure 4. Dimensions of the amide surrogate in 1 (neutral reduced
amide), 1′ (Z-protected reduced amide), 3 (semicarbazone fragment),
and 4 (semicarbazide fragment).
Figure 5. Crystal molecular conformations of the neutral reduced
dipeptide 1 (a), the Z-protected reduced dipeptide 1′ (b), and the reduced
azapeptide 4 (c). Major conformation in solution of the protonated
reduced peptide 1+ (d). The intramolecular hydrogen bonds are indicated
by broken lines.
Table 3. Main Torsional Angles (deg)a
reduced
peptides
reduced
azapeptide
iminoazapeptides
b
b
b
angle
1
1′
3
3′A
3′B
3′C
4
residue, in the gauche- or gauche+ conformation of the middle
reduced amide bond, and in the value of the “Pro”-"ψ" angle.
Both folded structures are intermediate between types I (trans
middle amide bond) and VI (cis middle amide bond) of the
peptide â-turn.24 The â-like structure of 1 also differs from
the crystal molecular structure of Boc-Proψ[CrH2NH]Leu-Gly-
NH2, where Leu-NH interacts with Boc-CO in a γ-like turn and
Gly-NH interacts with the reduced amide nitrogen to close a
five-membered cycle,8 similar to that observed for example in
the crystal structure of lidocaine Et2NCH2CONH(2,6-Me2Ph).25
The molecular conformation of the reduced azapeptide 4 (Figure
5c) is related partly to that of 1 (same conformation of the “Pro”
residue) and partly to that of Boc-Proψ[CrH2NH]Leu-Gly-NH2
(same five-membered cycle).8
It ensues that the neutral reduced amide acts in three different
ways: (i) as an intramolecular acceptor from the following
amide NH in Boc-Proψ[CrH2NH]Leu-Gly-NH2 (N‚‚‚N ) 2.65
Å) and in the reduced azapeptide 4 (N‚‚‚N ) 2.62 Å), (ii) as
an intramolecular donor to Boc-CO in Boc-Proψ[CrH2NH]Leu-
Gly-NH2 (N‚‚‚O ) 3.08 Å),8 and (iii) as an intermolecular donor
to Ala-CO in 1 (N‚‚‚O ) 3.02 Å) and to Piv-CO in 4 (N‚‚‚O )
3.14 Å) (Table 4).
ω0
175
180
173
-1
-2
-1
177
“Pro”
“φ1”
“ψ1”
“ω1”
“Ala”
“φ2”
“ψ2”
“ω2”
-80
158
-79
-93
72
96 -175
-73
-21
-72
134
176
-69
-68
-78
172
-70
135 -100
175
180
-70 -141 -178 -177 -176 -179
103
178 -178
128
-7
-177
41
-1
168
-6
-10
4
a The torsional angles are defined by considering the atoms of the
main chain, in a way similar to that in peptides.23 b Three independent
molecules per asymmetric unit.
atoms were replaced at 1.03 Å from N in the direction obtained by
refinement.21 Crystal data, fractional coordinates for the hydrogen and
non-hydrogen atoms, equivalent thermal parameters and anisotropic
temperature parameters for the non-hydrogen atoms, interatomic bond
lengths and bond angles, and torsional angles have been deposited as
Supporting Information.
Crystal Structures
CR f NR substitution has little influence on the dimensions
of the reduced amide bond in 1 and 4 (Figure 4). The sp3
character of the reduced amide nitrogen results in the same
staggered conformation of the CRCrNCR group ("ω" ) -70°
and -79°, respectively) and confers the same R-chirality on
the amine nitrogen. Acylation of the reduced amide nitrogen
in 1′ restores the sp2 character of the nitrogen, and the Cr-N
bond rotates by 175° in comparison with 1 (Figure 4). The
same fragment in the reduced analogue Boc-Proψ[CrH2NH]Leu-
Gly-NH2 of the oxytocin C-terminal tripeptide assumes a nearly
trans conformation (“ω” ) -157°) with a S-chiral sp3 nitrogen.8
A trans planar conformation is also observed for the imine group
in 3 and 3′ (Figure 4), but the CrdN and N-NR bonds are 0.1
Å shorter than the corresponding bonds in the peptides.22
The main conformational angles are listed in Table 3. The
two reduced peptides 1 and 1′ adopt a folded structure (Figure
5a,b) with an intramolecular (iPr)NH to Piv-CO hydrogen bond
(N‚‚‚O ) 2.94 and 2.85 Å, respectively) closing a 10-membered
cycle. However, they differ in the conformation of the Ala
In the iminoazapeptide series, due to the presence of three
independent molecules in the cell of 3′, four different molecular
structures have been found, but molecules 3′A and 3′B actually
adopt very similar structures (Table 3). In all cases, the
semicarbazone fragment is practically planar with a cis NNR-
CON system, in such a way that the C-terminal N-H bond is
directed toward the lone pair of the imine nitrogen (N...N )
2.60-2.65 Å) and that the only degree of freedom appears to
be the rotation of the “Pro” CR-Cr bond (Figure 6). The NRH
bond in 3′ is a proton donor to the C-terminal carbonyl of a
neighbor molecule (NRA‚‚‚OA ) 2.91 Å, NR ...OB ) 2.84 Å,
B
and NRC‚‚‚OA ) 2.91 Å) (Table 4).
Conformations in Solution
Before investigating the conformational properties of the
pseudopeptide analogues 1-4, we have considered the influence
(23) IUPAC-IUB Commission on Biochemical Nomenclature Eur. J.
Biochem. 1984, 138, 9-37.
(21) Taylor, R.; Kennard, O. Acta Crystallogr., Sect. B 1983, 39, 133-
138.
(22) Benedetti, E. In Chemistry and Biochemistry of Amino Acids,
Peptides and Proteins; Weinstein, B., Ed.; Marcel Dekker Inc.: New York,
1982; Vol. 6, pp 105-184.
(24) Rose, G. D.; Gierasch, L. M.; Smith, J. A. AdV. Protein Chem. 1985,
37, 1-109.
(25) Hanson, A. W.; Banner, D. W. Acta Crystallogr., Sect. B 1974, 30,
2486-2488.

Amide Surrogates
J. Am. Chem. Soc., Vol. 120, No. 37, 1998 9449
Table 4. Dimensions (Å, deg) of the Hydrogen Bonds
atoms
symmetry code
Reduced Peptide 1
N‚‚‚O/N
H‚‚‚O/N
N-H‚‚‚O/N
(iPr)NH to Piv-CO
Ala-NH to Piv-CO
2.94
3.14
1.93
2.14
166
162
-x, 1/2 + y, 1/2 - z
Reduced Peptide 1′
(iPr)NH to Piv-CO
(iPr)NH to AzAla-N
a
2.84
2.59
1.91
2.14
150
104
Iminoazapeptide 3
b
Iminoazapeptide 3′ c
NH2A to AzGly-NA
NH2B to AzGly-NB
NH2C to AzGly-NC
NH2A to Boc-COA
b
b
b
2.64
2.64
2.61
2.91
2.84
2.91
2.92
2.89
2.92
2.29
2.22
2.24
1.88
1.85
1.94
1.92
1.86
1.92
98
103
99
177
162
156
165
177
162
1 - x, y, z
1 + x, y, z
NH2B to Boc-COB
NH2C to AzGly-COA
AzGly-NRHA to AzGly-COB
AzGly-NRHB to AzGly-COB
AzGly-NRHC to AzGly-COB
1/2 + x, 7/2 - y, -z
-1/2 + x, 3/2 - y, -z
1/2 + x, 7/2 - y, -z
x, y, z
Reduced Azapeptide 4
(iPr)NH to AzAla-N
AzAla-NH to AzAla-CO
b
2.61
3.02
2.05
2.12
112
145
x, 1.5 - y, -0.5 + z
a Intramolecular hydrogen bond closing a 10-membered cycle b Intramolecular hyrogen bond closing a five-membered cycle c Three independent
molecules per asymmetric unit.
Figure 8. Influence of DMSO-d6 content in CDCl3/DMSO-d6 mixtures
Figure 6. Crystal molecular conformation of the iminoazadipeptides
on the C-terminal NH resonance (a) for the neutral reduced peptides
3′A (a), 3′C (b), and 3 (c). The intramolecular hydrogen bonds are
1, 1′M (major conformer), 1′m (minor conformer), the protonated
indicated by broken lines.
reduced peptide 1+, the methyleneoxypeptide 2, the iminoazapeptide
3, and the reduced azapeptide 4 and for the Ala-N+H2 resonances (b)
for 1+.
about 7. Therefore, under the physiological conditions, a
reduced peptide may assume a neutral or protonated form
depending on the environment. A previous study has shown
that protonation induces particular conformational properties in
dipeptides containing a C-terminal reduced amide bond.10a The
same probably holds true for 1 since the coupling constants in
the “Pro” CRHCrH2 system, which are related to the rotational
state of this bond, vary significantly with the pH (Figure 7).
In all four neutral pseudodipeptides 1-4, the small variation
of the (iPr)NH resonance with DMSO-d6 content in CDCl3/
DMSO-d6 mixtures (Figure 8) is typical of a solvent-protected
1
Figure 7. Influence of the pH on the H NMR data (chemical shifts
proton17b which is therefore intramolecularly hydrogen-bonded.
and coupling constants) for the “Pro” CRH-CrHAHB and AzAla-CâH3
This is corroborated by the low (iPr)NH stretching frequency
systems in the reduced peptide 1/1+ in water.
in CH2Cl2 (Table 4), inferior to the value expected for a free
of the pH in the 2-12 range on the 1H NMR data collected in
water. Only the resonances of 1 are significantly shifted at about
pH 6-8, while those of the other three derivatives are not
affected (Figure 7). It ensues that the semicarbazone and
semicarbazide groups are essentially neutral in the 2-12 pH
range, whereas the pKa value of the reduced amide bond in 1 is
vibrator (3425 cm-1).17b In DMSO, the (iPr)NH absorption is
shifted to a very low-frequency denoting its solvated state,
except for 3, where it retains the same frequency and sharp
profile as in CH2Cl2.
The Piv-CO frequency is practically independent of the
peptide bond surrogate and of the solvated (DMSO) or non-

9450 J. Am. Chem. Soc., Vol. 120, No. 37, 1998
Vanderesse et al.
Table 5. Amide NH and Piv-CO Stretching Frequencies for the Pseudodipeptides and their N- and C-Terminal Derivatives in Solutiona
compd
solvent
amide NH
Piv-CO
neutral reduced peptide
1
CH2Cl2
DMSO
CH2Cl2
DMSO
CH2Cl2
CH2Cl2
DMSO
CH2Cl2
DMSO
CH2Cl2
CH2Cl2
DMSO
CH2Cl2
CH2Cl2
DMSO
CH2Cl2
CH2Cl2
DMSO
CH2Cl2
3350b/3420w
3280
1611s
1613s
1610s
1611s
1N
1C
1+
3368b
protonated reduced peptide
3340m/3410m/3395m
3280b
1588s
1616s
1+N
1602m/1584m
1615s
1+C
2
3420s/3400w
3414s
methylenoxypeptide
iminoazapeptide
1614s
1615s
3263b
3411s
2C
3
3417s
1619s
1616s
3412s
3C
4
3414s
reduced azapeptide
3402s
1612s
1613s
3251b
3413
4C
a Broad and strong (b), sharp and strong (s), medium (m), and weak (w) absorptions. Values in bold characters denote intramolecularly hydrogen-
bonded vibrators.
Table 6. 1H NMR Data for the Peptide Surrogate in the Neutral 1
solvated (CH2Cl2) state of the (iPr)NH site, suggesting that the
and Protonated 1+ Reduced Dipeptides (MeCN-d3) and the Reduced
Azadipeptide 4 (CDCl3)
Piv-CO is not engaged in any intramolecular interaction. This
hypothesis is supported by the fact that a pseudopeptide and
the molecule reproducing its C-terminal part (namely 1/1C,
2/2C, 3/3C, and 4/4C) give very similar NH stretching
frequencies, independent of the presence of the Piv-CO.
Therefore, the only possible partner of the (iPr)NH is the peptide
surrogate itself, i.e. the sp3 nitrogen in 1 and 4, the ether oxygen
in 2, and the imine nitrogen in 3. This interaction closes a five-
membered pseudocycle as in the crystal structures of 3 and 3′
(Figure 6), 4 (Figure 5c), and Boc-Proψ[CrH2NH]Leu-Gly-
NH2.8 When not possible in the Z-containing reduced peptide
1′, the NH proton is more sensitive to DMSO solvation for both
cis and trans isomers of the Z-N amide bond (Figure 8).
Protonation of the reduced amide in 1+ induces noticeable
changes in the IR data (Table 4), especially for the Piv-CO
stretching frequency which shifts from 1616 to 1588 cm-1, a
low value typical of a Piv carbonyl engaged in a strong
interaction. No absorption expected for the free N+H2 group
when associated with PF6- in CH2Cl2 is observed at about 3245
cm-1, but a very broad and composite absorption at about 2800
cm-1 denotes the bonded character of the ammonium. The same
holds true when comparing the neutral and protonated deriva-
tives 1N and 1+N. We therefore conclude that the Piv carbonyl
and the ammonium in 1+ are hydrogen bonded in a γ-like folded
structure.10a
compd
1
1+
4
Chemical Shifts (ppm)
“Pro”-CRH
4.10
4.23
4.21
“Pro”-CrHAHB
2.67 (A)
2.32 (B)
1.80a
2.99
1.13
2.93 (A)
3.10 (B)
8.40a
3.59
1.38
3.12 (A)
2.62 (B)
3.59
“Ala”-NH or N+H2
“Ala”-CRH
“Ala”-CâH3
3.05
Coupling Constants (Hz)
3J(CRHCrHAHB)
3.8 (A)
8.3 (B)
-11.3
b
8.3 (A)
4.7 (A)
7.0 (B)
-11.3
5.0 (A)
7.4 (B)
1.6 (B)
-13.4
b
2J(CrHAHB)
3J(CrHAHBNH)
a Broad signal. b Nonmeasurable.
resonances are very sharp, by selective irradiation and chemical
exchange of the NH proton with water traces (Table 6). The
very small coupling constant of 1.6 Hz for 1+ indicates a rigid
conformation of the “Pro” CR-Cr bond placing the “Pro”-CRH
and “Pro”-CrHB protons in an orthogonal disposition corre-
sponding to "ψ1" ) 90° or 150°. Only the former value is
compatible with the γ-like folded conformation assumed by 1+
(Figure 5d). The vicinal coupling constants for the neutral
derivatives 1 and 4 denote the higher flexibility of the CRCrN
fragment than for 1+, in agreement with the IR evidence that
the only intramolecular interaction concerns the C-terminal
moiety in both derivatives.
The (iPr)NH absorption for 1+ is also modified by protonation
(Table 4) and the (iPr)NH proton is slightly more accessible to
DMSO solvation (Figure 8). The contribution at 3420 cm-1 in
CH2Cl2 is due to the free N-H vibrator, and the contribution
at 3400 cm-1, also present for 1+C, can be attributed to the
Conclusion
-
PF6 bonded amide NH.10a The component at 3340 cm-1
,
absent from 2C+, is typical of a hydrogen-bonded NH, and the
only possible partner is the Piv carbonyl. It results that
molecules 1+ are all folded by a N+H2 to Piv-CO interaction in
The neutral CRCrH2NHCR and CRCrH2NHNR fragments in 1
and 4, respectively, have quite similar dimensions. Moreover,
the reduced peptide 1, the methyleneoxypeptide 2, and the
reduced azapeptide 4 share the same conformational properties
in low polar solvents. Both nitrogen in 1 and 4 and the oxygen
in 2 act as an accepting group from the contiguous peptide NH
so as the intramolecular N-H‚‚‚N/O hydrogen bond closes a
five-membered pseudocycle. This conformation is not retained
in strong aprotic DMSO medium. There is no indication for
the existence in solution of the folded structure, related to the
â-turn in peptides with an i + 3 f i interaction closing a 10-
i
t
a γ-like turn and that part of them also present a PrNH to -
BuCO interaction in a â-like turn (Figure 5d). The high Piv-
CO stretching frequency in DMSO (Table 5) indicates that this
structure is not retained in DMSO, and the upfield-shifted Ala-
N+H2 resonances (Figure 8) confirm the solvated free character
of the ammonium group.
The “Pro” CRHCrHAHB spin system has been solved for 1
and 1+ in MeCN-d3 and for 4 in CDCl3, where the proton

Amide Surrogates
J. Am. Chem. Soc., Vol. 120, No. 37, 1998 9451
membered pseudocycle, which is observed in the solid state for
the reduced peptide 1. Although this â-like folded structure is
probably the consequence of the packing forces in the crystal,
it must be considered as potentially accessible to the reduced
peptide, methyleneoxypeptide, and reduced azapeptide motifs.
Contrary to the three above flexible peptide surrogates, the
semicarbazone in the iminoazapeptides 3 is a rigid fragment
assuming a planar cis structure with a N-H‚‚‚N hydrogen bond
closing a five-membered pseudocycle. This interaction is stable
enough to be retained in a strong solvating medium such as
DMSO. The only degree of freedom is then the rotation about
the single bonds contiguous to the semicarbazone motif.
Protonation of a reduced peptide induces noticeable confor-
mational changes due to the strong proton donating properties
of the ammonium group in 1+. The low pKa value of the
semicarbazide in 4, compared with the value of about 7 for the
reduced amide in 1, indicates that the former is not protonated
in water under the physiological conditions whereas the latter
is at least partly protonated. Therefore, the electronic state of
a reduced azapeptide is probably independent of the environ-
ment, whereas that of a reduced peptide is hardly predictable
and should depend on the nature of the neighboring groups.
Acknowledgment. The authors dedicate the present work
to the memory of D. Bayeul. The participation of L. David is
greatly acknowledged.
Supporting Information Available: Crystal data, fractional
coordinates for the hydrogen and non-hydrogen atoms, equiva-
lent thermal parameters and anisotropic temperature parameters
for the non-hydrogen atoms, interatomic bond lengths and bond
angles, and torsional angles (38 pages, print/PDF). See any
current masthead page for ordering information and Web access
instructions.
JA980937O