6786
J. Am. Chem. Soc. 1999, 121, 6786-6791
Mechanistic Studies of an Unusual Amide Bond Scission
Christopher J. Creighton, Todd T. Romoff,† Jane H. Bu, and Murray Goodman*
Contribution from the Department of Chemistry and Biochemistry, UniVersity of California at San Diego,
La Jolla, California 92093-0343
ReceiVed April 23, 1999
Abstract: Unusual acid cleavage reactions are reported for derivatives containing acylated N-methyl-R-
aminoisobutyryl (NMeAib) residues. The bond linking the NMeAib residue to the following amino acid is
cleaved. Through X-ray diffraction studies of the NMeAib containing molecules, we have shown that the
carbonyl oxygen atom of the preceding residue is in proximity to the carbonyl carbon of the NMeAib residue.
Thus, it can act as an internal nucleophile leading to a cleavage reaction by way of an oxazolinium ion
intermediate. Kinetic experiments for the cleavage reaction were carried out on a series of benzoyl dipeptide
derivatives (p-X-C6H4C(O)-NMeAib-Phe-OMe) where X is varied from NO2 to Cl. The value of F ) -1.335
for the Hammett linear free-energy relationship strongly supports the intermolecular oxazolinium intermediate
proposed.
Introduction
Results and Discussion
Recently, we reported1,2 an unusual reaction during the
deprotection of the cyclic hexapeptide somatostatin analogue
c[Phe-DTrp-Lys(Boc)-Thr(tBu)-Phe-NMeAib] (1) (where NMeAib
denotes an N-methyl-R-aminoisobutyryl residue). Upon treat-
ment of compound 1 with TFA and 1,2-ethanedithiol, two linear
deprotected hexapeptides were obtained: H-Phe-DTrp-Lys-Thr-
Phe-NMeAib-OH (2) and its C-terminal 2-thioethyl thioester
(3) (Figure 1). Since peptides are routinely treated with strong
acids such as TFA and anhydrous HF without loss of backbone
integrity, this facile amide bond cleavage3-9 is most unusual.
To investigate this amide bond cleavage reaction, we synthesized
peptides containing NMeAib residues. Crystallographic studies
were carried out on several of the NMeAib peptides. The crystal
structures were examined for possible steric evidence to explain
the lability of the amide bond between the NMeAib residue and
the adjacent amino acid in these peptides. No unusual bond
geometries were observed. The peptides were then subjected
to acidic conditions and their rates of acidolysis determined.
From these two studies, we concluded that the mechanism for
cleavage of this amide bond occurs via an oxazolinium ion
intermediate10,11 and not from the conventional AAC2 mecha-
nism.12,13
* To whom correspondence should be addressed.
† Current address: The Affymax Research Institute, Santa Clara,
California 95051.
(1) Spencer, J. R.; Delaet, N. G. J.; Toy-Palmer, A.; Antonenko, V. V.;
Goodman, M. J. Org. Chem. 1993, 58, 1635.
(2) Spencer, J. R.; Toy-Palmer, A.; Jiang, J. X.; Li, H.; Tran, T.-A.;
Romoff, T. Proc. 13th Am. Peptide Symp. 1994, Edmonton (Hodges, R.
S., Smith, John A., Eds.) pp 211-214, ESCOM, Leiden.
(3) Bender, M. L. Chem. ReV. 1960, 60, 53.
(4) Bromilow, J.; Abboud, J. L. M.; Lebrilla, C. B.; Taft, R. W.; Scorrano,
G.; Lucchini, V. J. Am. Chem. Soc. 1981, 103, 5448.
(5) Yates, K.; Modro, T. A. Acc. Chem. Res. 1978, 11, 190.
(6) Duffy, J. A.; Leinstein, J. A. J. Chem. Soc. 1960, 853.
(7) Williams, A. J. Am. Chem. Soc. 1976, 98, 5645.
(8) Perrin, C. L. Acc. Chem. Res. 1989, 22, 268.
(9) Edwards, J. T.; Derdall, G. D.; Wong, S. C. J. Am. Chem. Soc. 1978,
100, 7023.
Cleavage Studies using c[Phe-Ser(Bn)-Ser(Bn)-Phe-NMe-
Aib] (4). An investigation of the properties of cyclic pentapep-
tide 4 revealed that it also cleaves (Figure 2) specifically at the
NMeAib-Phe bond in neat TFA (t1/2 ) 2.0 h) to give the linear
pentapeptide H-Phe-Ser(Bn)-Ser(Bn)-Phe-NMeAib-OH (5).
We carried out kinetic experiments for the acidolysis of c[Phe-
Ser(Bn)-Ser(Bn)-Phe-NMeAib] (4). The TFA-mediated acidoly-
sis of c[Phe-Ser(Bn)-Ser(Bn)-Phe-NMeAib] is pseudo-first-order
with respect to the peptide. This is demonstrated by the linear
relationship shown in Figure 3B.
We also examined how solvent and acid concentrations
influence the rate of acidolysis (Table 1). The reaction rate
decreases by addition of water. Solvent polarity has a strong
influence on the rate of cleavage, as shown by acidolysis of 4
in 1:1 TFA:CH3CN (t1/2 ) 1.1 h) compared to cleavage in 1:1
TFA:CH2Cl2 (t1/2 ) 4.1 h). Increasing the concentration of TFA
in CH3CN increases the rate of scission.
Crystallographic Study of c[Phe-Ser(Bn)-Ser(Bn)-Phe-
NMeAib]. The ORTEP diagram of c[Phe-Ser(Bn)-Ser(Bn)-Phe-
NMeAib] (4) is shown in Figure 4. This structure demonstrates
that all of the amide bonds exhibit normal bond lengths and
geometries.14 The dihedral angles of the amide bonds range from
159.7 to -174.4°, and the C-N bond lengths range from 1.327
to 1.360 Å. The labile amide bond has a bond angle of -174.4°
and a bond length of 1.345 Å. We carried out 13C NMR studies
of c[Phe-Ser(Bn)-Ser(Bn)-Phe-NMeAib]. Our studies reveal no
unusual chemical shifts for the carbonyl carbons indicating all
amides exhibit normal geometries in solution. Thus, these results
indicate the sensitivity of the amide bond to acid does not arise
(12) March, J. Aliphatic Nucleophilic Substitution. In AdVanced Organic
Chemistry: Reactions, Mechanisms, and Structure, 4th ed.; Wiley &
Sons: New York, 1992; p 385.
(13) Lowery, T. H.; Richardson, K. S. Reactions of Carbonyl Compounds.
In Mechanism and Theory in Organic Chemistry, 3rd ed.; HarperCollins:
New York, 1987; p 720.
(10) Urban, J.; Vaisar, R. S.; Lee, M. S. Int. J. Pept. and Protein Res.
1996, 47, 1996.
(11) Proctor, P.; Gensmantel, N. P.; Page, M. I. J. Chem. Soc. Perkin
Trans. 2 1982, 1185.
(14) Ramachandran, G. N.; Sasisekharan, V. Conformation of Polypep-
tides and Proteins. In AdVances in Protein Chemistry; Anfinsen, C. B.,
Anson, M. L., Edsall, J. T., Richards, F. M., Eds.; Academic Press: New
York, 1968; pp 283-438.
10.1021/ja9913131 CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/14/1999