Sequencing hydroxyethylamine-containing peptides via Edman degradation

Article abstract of DOI:10.1021/ol026590i

(matrix presented) Hydroxyethylamine-containing peptides can be sequenced by automated Edman degradation to provide sequence information for peptide segments on either side of the peptide backbone modification.

Full text of DOI:10.1021/ol026590i

ORGANIC
LETTERS
2002
Vol. 4, No. 20
3469-3472
Sequencing
Hydroxyethylamine-Containing Peptides
via Edman Degradation
Matthias Brewer,† Thorsten Oost,† Chanokporn Sukonpan,‡
,†,‡
Michael Pereckas,§ and Daniel H Rich*
Department of Chemistry and School of Pharmacy, UniVersity of Wisconsin-Madison,
Madison, Wisconsin 53706, and The Protein and Nucleic Acid Shared Facility and
Department of Biochemistry, Medical College of Wisconsin,
Milwaukee, Wisconsin 53226
dhrich@facstaff.wisc.edu.
Received July 23, 2002
ABSTRACT
Hydroxyethylamine-containing peptides can be sequenced by automated Edman degradation to provide sequence information for peptide
segments on either side of the peptide backbone modification.
Modification of a peptide backbone through the incorporation
of peptide bond isosteres has proven to be a successful
approach to the development of potent enzyme inhibitors.1-3
Of these modifications, hydroxyethylamine4 dipeptide iso-
steres have found particular success in the development of
inhibitors for an array of enzymes including renin,5 HIV
protease,6-9 and more recently â- secretase.10,11 We have
observed that automated Edman degradation12 of peptides
containing the hydroxyethylamine moiety provides sequence
information for peptide segments on both sides of the peptide
backbone modification.We first noted this during Edman
degradation of epimeric hydroxyethylamines 1a and 1b,
which were prepared as potential substrate-based inhibitors3,13
of botulism neurotoxin-B metallopeptidase (BoNT-B).14 The
hydroxyethylamine moiety is known to inhibit the thermoly-
sin class of metallopeptidases.4
* To whom correspondence should be addressed at the Department of
Chemistry.
† Department of Chemistry.
‡ School of Pharmacy.
§ The Protein and Nucleic Acid Shared Facility.
(1) Leung, D.; Abbenante, G.; Fairlie, D. P. J. Med. Chem. 2000, 43,
305.
The desired hydroxyethylamines 1a/b were synthe-
sized by on-resin reductive amination of the resin-bound
(2) Babine, R. E.; Bender, S. L. Chem. ReV. 1997, 97, 1359.
(3) Rich, D. H. In ComprehensiVe Medicinal Chemistry. The Rational
Design, Mechanistic Study and Theraputic Application of Chemical
Compounds; Hansh, C. S., Sammes, P. G., Taylor, J. B., Eds.; Pergamon
Press: New York, 1990; p 391.
(4) (a) Gordon, E. M.; Godfrey, J. D.; Pluscec, J.; Vonlangen, D.;
Natarajan, S. Biochem. Biophys. Res. Commun. 1985, 126, 419. (b) Godfrey,
J. D.; Gordon, E. M.; Von Langen, D.; Engebrecht, J.; Pluscec, J. J. Org.
Chem. 1986, 51, 3073.
10.1021/ol026590i CCC: $22.00 © 2002 American Chemical Society
Published on Web 09/04/2002

C-terminal sequence with an epimeric mixture of 1-ethoxy
ethyl (EE)-protected R-hydroxy aldehyde 2a/b.15 Chain
One report by Jo¨rnvall et al.17 documents a similar
observation for Edman degradation of reduced amide con-
taining peptide 11. To test the generality of their observation,
elongation,16 followed by final deprotection and cleavage
from resin provided a diastereomeric mixture of hydroxy-
ethylamine analogues 1a/b, which were readily separable by
preparative reverse-phase HPLC. Although mass spectrom-
etry indicated the desired product, the lower than expected
biological activity of these compounds prompted us to more
fully characterize them to rule out the possibility that
rearrangements had occurred during peptide synthesis.
As a means of characterization, hydroxyethylamines 1a
and 1b were submitted for protein sequencing via automated
Edman degradation. We expected to observe the N-terminal
residues up to the point of modification. However, degrada-
tion provided both the expected N-terminal sequence, and
also a sequence commencing after the dipeptide isostere
(Table 1). To rule out sample decomposition as the cause of
we prepared reduced amides 12, 13,18 and 14.19 Reduced
amide 12 was synthesized by on-resin reductive amination20
of aldehyde Fmoc-Gln(Trt)-H15 with the amino group of the
resin-bound C-terminal sequence.16 Chain elongation,16 fol-
lowed by final deprotection and cleavage from resin,
provided the desired reduced amide 12, which was purified
by preparative reverse-phase HPLC. Reduced amides 13 and
14 were synthesized in a similar fashion. Degradation again
provided both the N-terminal sequence and a secondary
sequence starting adjacent to the reduced amide modification
(Table 1).
Table 1. Peptide Sequencing Resultsa
1a
1b
12
13
14
15
sequencing
step
Nb Cc
N
C
E
N
C
N
C
N
C
N
C
1
2
3
4
5
6
7
8
L
S
E
L
E
T
S
L
S
E
L
L
S
E
L
E
T
S
E
S
E
L
S
E
L
N
K
T
R
A
N
Q
R
A
T
N
K
T
R
A
N
Q
R
S
A
A
A
K
L
D
D
R
A
D
A
L
Q
A
G
A
D
D
R
A
D
A
L
Q
A
G
A
A
K
L
K
R
K
Y
W
W
D
D
R
A
D
A
L
Q
A
G
A
D
D
R
A
D
A
L
Q
A
G
A
K
L
K
R
K
Y
W
W
K
N
9
10
11
12
13
14
15
Y
a Sequencing was performed on a Beckman Coulter Porton LF-3000
sequencer using the standard sequencing program. b N ) observed sequence
commencing at the N-terminus of the peptide. c C ) observed sequence
commencing after peptide backbone modification.
Edman degradation of R-hydroxy amide 15,15 however,
provided only the expected N-terminal sequence up to the
this result, MALDI-TOF mass spectrometry was performed
on each sample. In each case only the full-length peptides
were observed.
(6) Miller, M.; Schneider, J.; Sathyanarayana, B. K.; Toth, M. V.;
Marshall, G. R.; Clawson, L.; Selk, L.; Kent, S. B. H.; Wlodawer, A. Science
1989, 246, 1149.
(7) Rich, D. H.; Green, J.; Toth, M. V.; Marshall, G. R.; Kent, S. B. H.
J. Med. Chem. 1990, 33, 1285.
(5) (a) Ryono, D. E.; Free, C. A.; Neubeck, R.; Samaniego, S. G.;
Godfrey, J. D.; Petrillo, E. W., Jr. Proc. Am. Pept. Symp., 9th. 1985, 739.
(b) Dann, J. G.; Stammers, D. K.; Harris, C. J.; Arrowsmith, R. J.; Davies,
D. E.; Hardy, G. W.; Morton, J. A. Biochem. Biophys. Res. Commun. 1986,
134, 71. (c) Cooper, J. B.; Foundling, S. I.; Blundell, T.; Arrowsmith, R.
J., Harris, C. J.; Champness; Topics in Medical Chemistry, Fourth SCI-
RSC Medicinal Chemistry Symposium; Leening, P. R., Ed.; Special Pub.
No. 65, The Royal Society of Chemistry: London, 1988; p 309.
(8) Roberts, N. A.; Martin, J. A.; Kinchington, D.; Broadhurst, A. V.;
Craig, J. C.; Duncan, I. B.; Galpin, S. A.; Handa, B. K.; Kay, J.; Krohn,
A.; Lambert, R. W.; Merrett, J. H.; Mills, J. S.; Parkes, K. E. B.; Redshaw,
S.; Ritchie, A. J.; Taylor, D. L.; Thomas, G. J.; Machin, P. J. Science 1990,
248, 358.
(9) Lebon, F.; Ledecq, M. Curr. Med. Chem. 2000, 7, 455-477.
(10) Wolfe, M. S.; Selkoe, D. J. In PCT Int. Appl.; The Brigham and
Women’s Hospital, Inc., USA; WO 0214264 A2, 2002, p 59.
3470
Org. Lett., Vol. 4, No. 20, 2002

Scheme 1
point of modification (Table 1). Although hydroxy amide
thiourea to liberate the C-terminal peptide fragment while
still in the presence of phenyl isothiocyanate.17 As shown in
Scheme 1, phenyl isothiocyanate reacts with secondary amine
1 to provide bis-thiourea 3. The internal thiourea of 3 cyclizes
under the reaction conditions to provide phenylthiohydantoin
4 and C-terminal peptide 5. Peptide 5 reacts with another
equivalent of phenyl isothiocyanate to provide thiourea 6.
Acid-induced cyclization and cleavage of the primary thio-
ureas provides the expected phenylthiohydantoins 7 and 9
and peptide fragments 8 and 10 available for the next round
of sequencing. Formation of bis-thiourea 3 is not quantitative;
15 has a secondary alcohol that could be a site for acylation
by phenyl isothiocyanate, no chain cleavage was observed.
This indicates the key role played by the secondary amine
present in the reduced amide and hydroxyethylamine deriva-
tives during the degradation process.21 We hypothesize that
under Edman degradation conditions the secondary amine
of the hydroxyethylamine reacts in a fashion similar to the
N-terminal primary amine and thus provides a second site
for peptide bond cleavage. It should be noted that for each
of the hydroxyethylamine and reduced amide substrates
examined the first residue of the C-terminal sequence
(glutamic acid, Table 1, row 1, columns labeled C-term) was
observed during the first round of degradation. This was also
noted by Jo¨rnvall for reduced amide 11 and can be explained
by spontaneous cyclization and cleavage of the internal
(16) Peptides were synthesized on a Pioneer Peptide Synthesis System
using standard Fmoc protocol. For reviews on the practice of solid-phase
peptide synthesis, see: (a) Fields, G. B.; Lauer-Fields, J. L.; Liu, R.-q.;
Barany, G. In Synthetic Peptides: A User’s Guide; Grant, G. A., Ed.; Oxford
University Press: New York, NY, 2002. (b) Atherton, E.; Sheppard, R. C.
Solid-Phase Peptide Synthesis: A Practical Approach; IRL Press: Oxford,
U.K., 1989.
(11) Beck, J. P.; Gailunas, A.; Hom, R.; Jagodzinska, B.; John, V.;
Maillaird, M. In PCT Int. Appl.; Elan Pharmaceuticals, Inc., USA.;
Pharmacia + Upjohn Company; WO 0202518 A2, 2002, p 286.
(12) (a) Edman, P. Acta Chem. Scand. 1950, 4, 283. (b) Niall, H. D.
Methods Enzymol. 1973, 27, 942.
(13) Oost, T. K.; Sukonpan, C.; Rich, D. H. Proc. Am. Pept. Symp., 16th.
1999, 24.
(14) For a review of BoNT structure and function, see: Montecucco,
C.; Schiavo, G. Q. ReV. Biophys. 1995, 28, 423.
(15) Manuscript in preparation.
(17) Hempel, J.; Nilsson, K.; Larsson, K.; Jornvall, H. FEBS Lett. 1986,
194, 333. This reference was particularly difficult to locate because the
terminology “reduced amide” was not used in the text or abstract.
(18) MALDI-MS: calcd for [C81H147N29O26]+ 1943.1150, found 1943.1.
(19) MALDI-MS: calcd for [C83H154N29O24]+ 1941.1722, found 1941.2.
(20) Sasaki, Y.; Coy, D. H. Peptides 1987, 8, 119.
(21) For an examples of secondary amines participating in Edman
degradation, see: (a) Miranda, L. P.; Meldal, M. Angew. Chem., Int. Ed.
2001, 40, 3655. (b) Boeijen, A.; Liskamp, R. M. J. Tetrahedron Lett. 1998,
39, 3589.
Org. Lett., Vol. 4, No. 20, 2002
3471

depending on the amino acid sequence, some secondary
amine remains and undergoes cleavage in the next cycle of
Edman degradation.
The discovery that Edman degradation chemistry can be
used to sequence both the N-terminal and C-terminal
segments of hydroxyethylamine-derived peptides expands the
utility of this important reaction. This finding should be
useful for characterizing an important class of compounds
often used in the development of enzyme inhibitors.
aid in the Edman sequencing. This work was supported by
a research grant from NIHGMS (GM 59 956). T.K.O.
received postdoctoral fellowships from Fonds der Chemis-
chen Industrie and Deutsche Forschungsgemeinschaft. C.S.
received funding from the Royal Thai Government.
Supporting Information Available: Experimental pro-
cedure for the synthesis of compound 12. This material is
available free of charge via the Internet at http://pubs.acs.org.
Acknowledgment. We thank Dr. Martha Vestling for
obtaining the mass spectra, and Dr. Bassam T. Wakim for
OL026590I
3472
Org. Lett., Vol. 4, No. 20, 2002