Aza-amino acid scan for rapid identification of secondary structure based on the application of N-Boc-aza1-dipeptides in peptide synthesis

Article abstract of DOI:10.1021/ja039643f

Azapeptides, peptide analogues in which the α-carbon of one or more of the amino acid residues is replaced with a nitrogen atom, exhibit propensity for adopting β-turn conformations. A general protocol for the synthesis of azapeptides without racemization on solid phase has now been developed by introducing the aza-amino acid residue as an N-Boc-aza¹-dipeptide. This approach has been validated by the synthesis of six N-Boc-aza ¹-dipeptides and their subsequent introduction into analogues of the C-terminal peptide fragment of the human calcitonin gene-related peptide (hCGRP). By performing an aza-amino acid scan of such antagonist peptides, a set of aza-hCGRP analogues was synthesized to examine the relationship between turn secondary structure and biological activity.

Full text of DOI:10.1021/ja039643f

Published on Web 05/07/2004
Aza-Amino Acid Scan for Rapid Identification of Secondary
Structure Based on the Application of N-Boc-Aza1-Dipeptides
in Peptide Synthesis
Rosa E. Melendez and William D. Lubell*
Contribution from the De´partement de Chimie, UniVersite´ de Montre´al,
C.P. 6128, Succursale Centre-Ville, Montre´al, Que´bec H3C 3J7, Canada
Received November 17, 2003; E-mail: lubell@chimie.umontreal.ca
Abstract: Azapeptides, peptide analogues in which the R-carbon of one or more of the amino acid residues
is replaced with a nitrogen atom, exhibit propensity for adopting â-turn conformations. A general protocol
for the synthesis of azapeptides without racemization on solid phase has now been developed by introducing
the aza-amino acid residue as an N-Boc-aza1-dipeptide. This approach has been validated by the synthesis
of six N-Boc-aza1-dipeptides and their subsequent introduction into analogues of the C-terminal peptide
fragment of the human calcitonin gene-related peptide (hCGRP). By performing an aza-amino acid scan
of such antagonist peptides, a set of aza-hCGRP analogues was synthesized to examine the relationship
between turn secondary structure and biological activity.
Introduction
Promising leads for drug discovery, natural peptides suffer
from rapid metabolism, poor bioavailability, and short duration
of action that compromise their application as drugs in clinic.1-5
Peptide mimics that do not possess such shortcomings have been
prepared by modification of the side chain and the backbone
of native peptides as well as by the application of secondary
structure surrogates.6-15 New strategies for effectively analyzing
peptide structures to identify biologically active conformations
are needed to accelerate the transition from peptide to mimic.
Traditional approaches for studying peptide structure-activity
relationships feature typically the sequential replacement of the
amino acids in the peptide chain with alternative amino acids
to assess their importance for biological activity. For example,
replacement of an amino acid residue in the lead sequence by
either alanine, its enantiomer, or proline may respectively reveal
Figure 1. Schematic representation of a peptide and an azapeptide.
the significance of side chains, configuration, and conformation
for activity.16 Related scanning techniques have recently been
described involving systematic introduction of N-alkylamino
acids into peptides to ascertain the importance of amide protons
in hydrogen-bond interactions.17-20 By employing chemical
approaches for systematically modifying a peptide sequence with
structural constraints that favor particular geometry, new generic
tools may be developed for identifying active conformers
involved in peptide-receptor interactions.
Azapeptides possess one or more amino acids in which the
central R-carbon is replaced by nitrogen (Figure 1).21 Electronic
repulsion between the adjacent nitrogen atoms has been sug-
gested to account for the type I and II â-turn geometry that
aza-amino acid residues adopt in peptides, as predicted by
computation22-24 and observed by spectroscopic25,26 and crystal-
(1) Steer, D. L.; Lew, R. A.; Perlmutter, P.; Smith, A. I.; Aguilar, M.-I. Curr.
Med. Chem. 2002, 9, 811.
(2) Perez, J. J.; Corcho, F.; Llorens, O. Curr. Med. Chem. 2002, 9, 2209.
(3) Bursavich, M. G.; Rich, D. H. J. Med. Chem. 2002, 45, 541.
(4) Dougherty, J. M.; Probst, D. A.; Robinson, R. E.; Moore, J. D.; Klein, T.
A.; Snelgrove, K. A.; Hanson, P. R. Tetrahedron 2000, 56, 9781.
(5) Hruby, V. J.; Balse, P. M. Curr. Med. Chem. 2000, 7, 945.
(6) Hanessian, S.; McNaughton-Smith, G.; Lombart, H.-G.; Lubell, W. D.
Tetrahedron 1997, 53, 12789.
(7) Goodman, M.; Zhang, J. Chemtracts 1997, 10, 629.
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1999, 38, 1223.
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Am. Chem. Soc. 1992, 114, 6568.
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Adams, C. L.; Sundaram, A.; Jacobs, J. W.; Schultz, P. Science 1994, 261,
1303.
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34, 907.
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(16) Marshall, G. R. Tetrahedron 1993, 49, 3547.
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6187.
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(20) Takeuchi, Y.; Marshall, G. R. J. Am. Chem. Soc. 1998, 120, 5363.
(21) Gante, J. Synthesis 1989, 405.
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63.
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A R T I C L E S
Melendez and Lubell
Scheme 1. Synthesis of Azapeptides on Solid Phase Is Usually Accompanied by Hydantoin Formation33
lographic methods.27-30 Although controversy remains over the
role of aza-amino acids in â-turn formation, X-ray crystal
structures of linear, L-proline-containing azapeptide analogues
have revealed type I and II â-turns in which the aza-amino acid
residue is situated, respectively, at the i + 1 and i + 2 position
and the R-nitrogen assumes a pyramidal structure in all but one
structure.27,28 Aza-dipeptides and aza-tripeptides have also been
studied by NMR spectroscopy in solvents of varying polarity
using DMSO to disrupt hydrogen bonding.23,25 These studies,
combined with data from IR experiments and ab initio calcula-
tions,22 all have led to the accepted model in which noncyclic
aza-amino acids adopt either the i + 1 or i + 2 position in an
intramolecularly hydrogen-bonded â-turn structure.25 For ex-
ample, the aza-tripeptide Boc-Phe-azaLeu-Ala-OMe was shown
by NMR and IR spectroscopy to prefer a type II â-turn geometry
in which the aza-amino acid adopted the i + 2 position.23 Cyclic
aza-amino acid residues, such as azaPro and azaPip, exhibit high
cis-amide isomer populations N-terminal to the aza-residue in
peptides and type VI â-turn geometry as observed by NMR
and IR spectroscopy and X-ray analysis.26,29-32 Energetically
stable cis-peptide bonds have also been predicted to be favored
in studies of azaGly containing peptides by ab initio and DFT
methods.24 These studies indicate that systematic replacement
of the amino acids in a peptide sequence by their aza-amino
acid counterpart could serve as a general means for scanning
for bioactive â-turn conformations.
by the use of the N-2-hydroxy-4-methoxybenzyl group as
reversible amide protection, this method requires two additional
synthesis steps and employs less reactive carbamate instead of
isocyanate intermediates.37
Application of azapeptides in the study of biologically active
peptides has had significant success. For example, azapeptides
have exhibited longer duration of action relative to natural
peptides, presumably because of increased resistance to pro-
teases36,38 and because ureas are generally more chemically
stable than amides.39 The azaVal analogue of bovine angiotensin
II exhibited increased duration of pressor action relative to its
Val counterpart.40 Azapeptides have also been synthesized and
evaluated as inhibitors of cysteine41-44 and serine39,45 proteases.
Azapeptide analogues of peptide hormones have been synthe-
sized, including thyrotropin-releasing hormone (TRH),46 oxy-
tocin,47 eledoisin,48 enkephalin,49 and luliberin (LHRH).50 An
example of the latter, Zoladex is used clinically for the treatment
of prostate cancer.51 To enhance the application of azapeptides
in peptide science and medicinal chemistry, we present now a
general strategy for solid-phase azapeptide synthesis, suitable
for aza-amino acid scanning, based on the synthesis of N-Boc-
aza1-dipeptides and their introduction into peptides.
Results and Discussion
N-Boc-aza1-dipeptides were synthesized by the reaction of
N-Boc-N′-alkyl hydrazines with isocyanates derived from
R-amino benzyl esters and subsequent hydrogenolytic ester
cleavage (Scheme 2).
The synthesis of N-protected N′-substituted hydrazines has
been the subject of much research, and different methodologies
have proven effective for different N′-substituents.55 N-Boc-
protected hydrazine derivatives have been prepared for making
aza-analogues of most of the proteinogenic amino acids.52 We
Azapeptide synthesis has, however, been challenging, requir-
ing a combination of hydrazine34,35 and peptide chemistry.33,36
Typically, an N-protected N′-substituted hydrazine is reacted
with a “carbonyl donor” such as an isocyanate or an active
carbamate.21 A significant drawback in the implementation of
these protocols on solid phase has been intramolecular hydantoin
formation of the resin-bound isocyanate or active carbamate
intermediate prior to reaction with the substituted hydrazine
(Scheme 1).33 Although hydantoin formation may be prevented
(37) Liley, M.; Johnson, T. Tetrahedron Lett. 2000, 41, 3983.
(38) Din, H.; Gray, C. J.; Ireson, J. C.; McDonald, R. J. Chem. Technol.
Biotechnol. 1991, 50, 181.
(39) Gassman, J. M.; Magrath, J. Bioorg. Med. Chem. Lett. 1996, 6, 1771.
(40) Hess, H.-J.; Moreland, W. T.; Laubach, G. D. J. Am. Chem. Soc. 1963, 85,
4040.
(41) Xing, R.; Hanzlik, R. P. J. Med. Chem. 1998, 41, 1344.
(42) Graybill, T. L.; Ross, M. J.; Gauvin, B. R.; Gregory, J. S.; Harris, A. L.;
Ator, M. A.; Rinker, J. M.; Dolle, R. E. Bioorg. Med. Chem. Lett. 1992, 2,
1375.
(43) Graybill, T. L.; Dolle, R. E.; Helaszek, C. T.; Ator, M. A.; Strasters, J.
Bioorg. Med. Chem. Lett. 1995, 5, 1197.
(44) Magrath, J.; Abeles, R. H. J. Med. Chem. 1992, 35, 4279.
(45) Semple, J. E.; Rowley, D. C.; Brunck, T. K.; Ripka, W. C. Bioorg. Med.
Chem. Lett. 1997, 7, 315.
(46) Zhang, W.-J.; Berglund, A.; Kao, J. L.-F.; Couty, J.-P.; Gershengorn, M.
C.; Marshall, G. R. J. Am. Chem. Soc. 2003, 125, 1221.
(47) Niedrich, H. J. Prakt. Chem. 1972, 314, 769.
(48) Oehme, P.; Bergmann, J.; Falck, M.; Reich, J. G.; Vogt, W. E.; Niedrich,
H.; Pirrwitz, J.; Berseck, C.; Jung, F. Acta Biol. Med. Ger. 1972, 28, 109.
(49) Han, H.; Yoon, J.; Janda, K. D. Bioorg. Med. Chem. Lett. 1998, 8, 117.
(50) Dutta, A. S.; Furr, B. J. A.; Giles, M. B.; Valcaccia, B. J. Med. Chem.
1978, 21, 1018.
(51) Furr, B. J. A.; Valcaccia, B. E.; Hutchinson, F. G. Br. J. Cancer 1983, 48,
140.
(25) Andre´, F.; Vincherat, A.; Boussard, G.; Aubry, A.; Marraud, M. J. Pept.
Res. 1997, 50, 372.
(26) Zouikri, M.; Vicherat, A.; Aubry, A.; Marraud, M.; Boussard, G. J. Pept.
Res. 1998, 52, 19.
(27) Andre´, F.; Boussard, G.; Bayeul, D.; Didierjean, C.; Aubry, A.; Marraud,
M. J. Pept. Res. 1997, 49, 556.
(28) Benatalah, Z.; Aubry, A.; Boussard, G.; Marraud, M. Int. J. Pept. Protein
Res. 1991, 38, 603.
(29) Lecoq, A.; Boussard, G.; Marraud, M. Tetrahedron Lett. 1992, 33, 5209.
(30) Lecoq, A.; Boussard, G.; Marraud, M.; Aubry, A. Biopolymers 1993, 33,
1051.
(31) Didierjean, C.; Aubry, A.; Wyckaert, F.; Boussard, G. J. Pept. Res. 2000,
55, 308.
(32) Hemmerlin, C.; Cung, M. T.; Boussard, G. Tetrahedron Lett. 2001, 42,
5009.
(33) Quibell, M.; Turnell, W. G.; Johnson, T. J. Chem. Soc., Perkin Trans. 1
1993, 2843.
(34) Biel, J. H.; Drukker, A. E.; Mitchell, T. F.; Sprengeler, E. P.; Nuhfer, P.
A.; Conway, A. C.; Horita, A. J. Am. Chem. Soc. 1959, 81, 2805.
(35) Brosse, N.; Pinto, M.-F.; Jamart-Gre´goire, B. J. Org. Chem. 2000, 65, 4370.
(36) Gray, C. J.; Quibell, M.; Baggett, N.; Hammerle, T. Int. J. Pept. Protein
Res. 1992, 40, 351.
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N-Boc-Aza1-Dipeptides in Peptide Synthesis
A R T I C L E S
Scheme 2. N-Boc-Aza1-Dipeptide Strategy for Azapeptide Synthesis
Scheme 3. Synthesis of Substituted Hydrazines46,52-54
synthesized hydrazines to mimic four amino acid side-chains:
Phe, Val, Ala, and Pro. Condensation of tert-butyl carbazate 1
with the appropriate aldehyde or ketone gave an acyl hydrazone
that was reduced best by catalytic hydrogenation or hydride
addition if the substituent was, respectively, aromatic or
aliphatic. Benzyl- and isopropyl-substituted hydrazines 2 and
3 were isolated in 81% and 75% yields, respectively, using these
procedures.52,53 Attempts to synthesize azaAla precursor 4 by
condensation of carbazate 1 with formaldehyde followed by
reduction, as well as by alkylation of 1 with iodomethane, did
not render the desired product in our hands. Methyl hydrazine
4 was synthesized by alkylation of the corresponding triphenyl
phosphinimine with iodomethane in 53% yield.54 Pyrazolidine
5 was synthesized by acylation of carbazate 1 with benzylchlo-
roformate prior to alkylation with 1,3-dibromopropane and
removal of the Cbz group by hydrogenolysis over palladium-
on-carbon in methanol (Scheme 3).46
Amino ester isocyanates 6-9 were synthesized from the
corresponding L-amino benzyl ester hydrochloride salts using
a 1.93 M solution of phosgene in toluene containing 120 mol
% of pyridine at 0 °C for 2h.56 Isocyanates 6-9 were employed
immediately in reactions with hydrazines 1-5. All N-Boc-aza1-
dipeptide benzyl esters were obtained in excellent yields (90-
98%) after purification by silica gel chromatography, with the
exception of N-Boc-azaVal-Gly-OBn 12, which was isolated
in 34% yield. N-Boc-aza1-dipeptides 16-21 were finally
prepared by hydrogenolysis of benzyl esters 10-15 using
palladium-on-carbon in ethanol (Scheme 4).
Although carbamate N-protection has been shown to be
superior to amide N-protection at preventing racemization of
R-amino acids during peptide synthesis,58 to the best of our
knowledge, there have been no reports on the use of urea nor
semicarbazide-protected R-amino acids in peptide chemistry.
The configurational integrity of the C-terminal amino acid
residue during the synthesis and application N-Boc-aza1-
dipeptides was ascertained by the solution-phase synthesis of
aza-tripeptides N-Boc-azaPhe-Ala-Phe-OMe 22. N-Boc-azaPhe-
(L)Ala-OH 17 was coupled to both L- and D-HCl‚Phe-OMe
using TBTU, HOBt, and DIEA in acetonitrile (Scheme 5). The
diastereomeric purity of the resulting aza-tripeptides (S,S)- and
(S,R)-22 was measured by integration of the diastereotopic
methyl ester singlets at 3.20 and 3.26 ppm, respectively, in the
1H NMR spectrum taken in benzene-d6. Spectral analysis of
aza-tripeptide (S,S)-22 during incremental additions of (S,R)-
22 established that (S,S)-22 was of >99% purity (>99:1 d.r.),
the limits of detection. Azapeptides can thus be made in high
stereoisomeric purity.57
(52) Dutta, A. S.; Morley, J. S. J. Chem. Soc., Perkin Trans. 1 1975, 1712. To
our knowledge, no protocols have been reported for the synthesis of the
Boc-protected hydrazine analogues for making aza-analogues of Lys, Arg,
His, and Met; the synthesis of hydrazine analogues for aza-analogues of
Ser, Thr, and Cys is unfeasible because of their masked aldehyde nature.
(53) Calabretta, R.; Gallina, C.; Giordano, C. Synthesis 1991, 536.
(54) Tsˇubrik, O.; Ma¨eorg, U. Org. Lett. 2001, 3, 2297.
Solution-phase synthesis of 22 was examined because of the
presence of its sequence in a peptide antagonist of the potent
vasodilator, human calcitonin gene-related peptide (hCGRP, 23),
(57) Melendez, R. E.; Lubell, W. D. In Peptides 2002, Proceedings of the 25th
European Peptide Symposium; Benedetti, E., Ed.: Italy, 2002; p 204.
(58) Johansson, A.; Akerblom, E.; Ersmark, K.; Lindeberg, G.; Hallberg, A.
J. Comb. Chem. 2000, 2, 496 and refs cited therein.
(55) Ragnarsson, U. Chem. Soc. ReV. 2001, 30, 205.
(56) Nowick, J. S.; Powell, N. A.; Nguyen, T. M.; Noronha, G. J. Org. Chem.
1992, 57, 7364.
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A R T I C L E S
Melendez and Lubell
Scheme 4. Synthesis of N-Boc-Aza1-Dipeptides 16-21
Scheme 5. Coupling of N-Boc-Aza1-Dipeptides to Amino Acids Using Standard Peptide Conditions Is Achieved in Higher than 99 % d.e.57
Scheme 6. Synthesis of Peptides 24-29 on Solid Phase Using Oxime Resin
Table 1. Sequence of R-hCGRP (23), Peptide Antagonists 24 and 25, and Azapeptides 26-29
(23) R-hCGRP
ACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF−NH
2
compound
sequence
tR (min)a
formula
MS (calcd for [M + H]+)
HPLC purity
(24) [D31, P34, F35]CGRP29-37
(25) [D31, P34, F35]CGRP27-37
(26) [D31, P34, azaF35]CGRP29-37
(27) [D31, azaP34, F35]CGRP29-37
(28) [azaF27, D31, P34, F35]CGRP27-37
(29) [D31, P34, azaF35]CGRP27-37
PTDVGPFAF-NH2
FVPTDVGPFAF-NH2
PTDVGPFAF-NH2
PTDVGPFAF-NH2
FVPTDVGPFAF-NH2
FVPTDVGPFAF-NH2
13.0
15.1
13.2
12.7
17.0
14.3
C46H64N10O12
C60H82N12O14
C45H63N11O12
C45H63N11O12
C59H81N13O14
C59H81N13O14
949.4 (949.4)
1195.9 (1195.9)
950.5 (950.5)
950.5 (950.5)
1196.3 (1196.6)
1196.6 (1196.6)
92
>99
91
88
>99
92
a
20-80% acetonitrile in H2O containing 0.01% TFA over 20 min, in vol: 10 µL.
a 37-amino acid neuropeptide that is characterized by a disulfide
loop between residues 2-7, an R-helix between residues 8-18,
and an aminated C-terminus (Table 1).59 Structure activity
studies have found that C-terminus analogues of hCGRP, such
as peptides 24 and 25, exhibit antagonist activity and have been
suggested to adopt bioactive turn conformations.60 To explore
the power of aza-amino acid scanning, our synthetic approach
was used to make aza-analogues of the 9 and 11 residue peptide
antagonists 24 and 25. Because aza-amino acids have been
shown to adopt i + 1 and i + 2 positions of â-turns, azapeptides
26-29 were synthesized to study the importance of turn regions
about the aromatic residues for antagonist activity. Employing
aza1-dipeptide fragments, hydantoin formation was avoided
during azapeptide synthesis on solid phase. Azapeptides ana-
logues 26-29 were prepared for evaluation as potential hCGRP
antagonists (Table 1). Scheme 6 shows the synthesis of peptides
24-29 on solid phase using oxime resin.
(59) Breeze, A. L.; Harvey, T. S.; Bazzo, R.; Campbell, I. D. Biochemistry 1991,
30, 575.
(60) Carpenter, K. A.; Schmidt, R.; von Mentzer, B.; Haglund, U.; Roberts, E.;
Walpole, C. Biochemistry 2001, 40, 8317.
N-Boc-aza1-dipeptides 16, 17, and 20 were incorporated into
azapeptides 26-29 using a Boc protection strategy on oxime
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N-Boc-Aza1-Dipeptides in Peptide Synthesis
A R T I C L E S
resin.61 Removal of the Boc group was carried out using 25%
TFA in DCM (30 min) followed by neutralization in 10% DIEA
in DMF (10 min). Of particular note, the Boc removal from
the aza-amino acid required longer reaction times and was car-
ried out during peptide synthesis for 2 h instead of the 30 min
period necessary for performing the analogous deprotection with
natural amino acid residues. Peptide couplings were carried out
with 3 equiv each of the appropriate Boc-protected amino acid
or N-Boc-aza1-dipeptides, TBTU, HOBt, and DIEA, in DMF
for 2 h. When coupling to aza-amino acid residues, extended
reaction times (16 h) were employed between washing of the
resin and addition of fresh reagents, because acylation of aza-
amino acid residues had been reported to be slower than similar
couplings to ordinary amino acid residues.36 Final cleavage of
the peptide from the resin was performed using a 1:1 mixture
of saturated NH3 in MeOH/DCM. Removal of the side-chain-
protecting groups [Thr(OBn) and Asp(OBn)] was carried out
by hydrogenolysis first over Pd(OH)2, followed by hydrogeno-
lysis over Pd/C in ethanol. The Boc group of the final peptide
was removed using 25% TFA in DCM, and the peptides 24-
29 were purified by HPLC to furnish 10-20% yields based on
initial loading of 0.3 mmol/g. Characterization of the peptides
was carried out by LC-MS, and purity was assessed by UV
peak integration.
in absolute ethanol, treated with a suspension of 10 mol % of Pd/C
(10 wt %) in ethanol, and stirred under H2 at 1 atm for 30 min. The
reaction was filtered over Celite and the solvent was removed by rotary
evaporation to obtain N-Boc-aza1-dipeptide (16-21).
Boc-AzaPhe-L-Ala-OH (17, 51 mg, 70% yield) was sublimed at
50-55 °C; Rf ) 0.16 (10% MeOH in DCM); [R]20D 3.9° (c 1.0, MeOH);
1H NMR 300 MHz (CD3OD): δ 7.34-7.23 (m, 5H), 4.22 (q, 1H,
J ) 7 Hz), 1.48 (s, 9H), 1.39 (d, 3H, J ) 7 Hz); 13C NMR 75 MHz
(CD3OD): δ 176.0, 158.8, 155.6, 136.9, 129.0, 128.5, 127.6, 81.5, 50.9,
49.7, 27.5, 17.8; HRMS (FAB) m/e: 338.1716 (calcd for C16H24N3O5
(M + H+), 338.1710).
Boc-AzaPhe-L-Ala-L-Phe-OCH3 [(S,S)-22]. Aza-dipeptide 17 (78
mg, 0.23 mmol) was dissolved in 1 mL of acetonitrile, cooled to 0 °C,
treated with TBTU (74 mg, 0.23 mmol) and HOBt (31 mg, 0.23 mmol),
stirred at 0 °C for 10 min, and treated with a solution of L-phenylalanine
methyl ester hydrochloride (99 mg, 0.45 mmol) and DIEA (89 mg,
0.69 mmol) in 0.2 mL of acetonitrile. After being stirred for 12 h, the
solvent was evaporated, and the crude product was dissolved in DCM
and extracted with small aliquots of concentrated NaHCO3 solution, 1
M NaH2PO4, and brine. The solvent was evaporated to collect pure
(S,S)-22 (93 mg, 81% yield): mp ) 120-122 °C; [R]20D 37.8° (c 1.0,
CHCl3); Rf ) 0.75 (10% MeOH in DCM); 1H NMR (400 MHz,
C6D6): δ 7.26-7.04 (m, Ar, 10H) 6.12 (d, J ) 7 Hz, NH, 1H), 6.02
(br s, NH, 1H), 5.01 (dd, J ) 6 and 14 Hz, 1H), 4.59 (m, 1H), 3.20 (s,
3H), 3.16 (dd, J ) 6 and 14 Hz, 1H), 3.05 (dd, J ) 6 and 14 Hz, 1H),
3.32 (d, J ) 7 Hz, 3H), 1.25 (s, 9H); 13C NMR 75 MHz (CDCl3): δ
172.7, 171.9, 157.4, 154.4, 136.1, 136.0, 129.5, 129.1, 129.0, 128.8,
128.2, 127.3, 82.7, 53.6, 52.6, 50.8, 50.1, 38.0, 28.3, 18.8; ESI/MS
m/z: 521.2, 100% (calcd. for C26H34N4O6Na (M + Na+) 521.2). Boc-
AzaPhe-L-Ala-D-Phe-OCH3 [(S,R)-22] was synthesized from 17 and
D-phenylalanine methyl ester hydrochloride according to the procedure
Conclusion
N-Boc-azadipeptides containing different aza-amino acids
(azaGly, azaVal, azaPhe, azaAla, and azaPro) have been
synthesized in solution and demonstrated to be configurationally
stable building blocks for solution and solid-phase peptide
synthesis. The utility of this methodology was illustrated by
the solid-phase synthesis of azapeptides 26-29 possessing 9
and 11 residues using standard peptide coupling conditions on
oxime resin. We are currently working with collaborators to
evaluate the biological activity of azapeptides 26-29 as potential
hCGRP antagonists.
described above (83% yield); [R]20 -5.8° (c 2.0, CHCl3); Rf ) 0.66
D
1
(10% MeOH in DCM); H NMR (300 MHz C6D6): δ 7.22-7.00 (m,
Ar, 10H), 5.87 (d, J ) 6 Hz, NH, 1H), 5.72 (s, NH, 1H), 5.02 (dd, J
) 8 and 14 Hz, 1H), 4.52 (m, 1H), 3.26 (s, 3H), 3.12 (dd, J ) 6 and
14 Hz, 1H), 2.95 (dd, J ) 8 and 14 Hz, 1H), 1.28 (s, 9H), 1.18 (d, J
) 7 Hz, 3H); 13C NMR 75 MHz (CDCl3): δ 172.7, 172.0, 157.3, 154.5,
136.2, 136.1, 129.4, 129.0, 128.9, 128.7, 128.6, 128.0, 127.2, 82.6,
53.5, 52.4, 49.9, 38.7, 37.9, 28.2; ESI/MS m/z: 521.2, 100%, (calcd.
for C26H34N4O6Na (M + Na+) 521.2).
Peptide Synthesis (24-29). Peptide synthesis was performed in an
automated shaker using oxime resin.61 Aspartic acid and threonine were
introduced as Boc-L-Asp(Bn)-OH and Boc-L-Thr(Bn)-OH. Couplings
were performed with Boc-protected amino acids (300 mol %) and
N-Boc-aza1-dipeptides (16, 17, and 20, 300 mol %), respectively, with
TBTU (300 mol %), HOBt (300 mol %), and DIEA (300 mol %) in
DMF for 2 h. The resin was agitated with N2 bubbles during the
coupling, rinsing, and deprotection sequences. Coupling reactions were
monitored by the Kaiser ninhydrin test.62 In cases of incomplete
couplings, the resin was resubmitted to the same coupling conditions.
Deprotections were performed with 25% TFA in DCM (30 min), and
the resin was neutralized with 10% DIEA in DMF (10 min). The
peptides were obtained by cleavage from the resin with a 1:1 solution
of saturated NH3 in MeOH/DCM (30 min). The crude product was
purified by LC-MS and then submitted to hydrogenolysis over 10 mol
% Pd(OH)2 in EtOH for 16 h at 7 atm and then 10 mol % Pd/C in
EtOH at 1 atm for 30 min, followed by Boc deprotection using 1:4
TFA/DCM for 30 min. The final peptides (24-29) were purified with
semipreparative LC-MS (Previal C18 column, 22 × 250 mm2, particle
size 5 µm) with solvent A, H2O (0.01% TFA), and solvent B,
acetonitrile (0.01% TFA), using a gradient of 20-80% over 20 min at
a flow rate of 15 mL/min. Retention times (tR) are reported in minutes.
PTDVGPFAF-NH2 (24). Purity 92% by LC-MS (tR ) 13.1);
LRMS calcd for C46H65N10O12 (M + H+), 949.5; found, 949.4.
Experimental Section
General Data. Solvents and reagents were purified as specified in
the Supporting Information. Mass spectral data and HRMS was obtained
by the Universite´ de Montre´al Mass Spectrometry facility.
General Protocol for the Synthesis of N-Boc-Aza1-Dipeptide
Benzyl Esters (10-15). L-Amino acid benzyl ester hydrochloride (1
equiv) and pyridine (4 equiv) were dissolved in DCM, cooled to 0 °C,
treated with a 1.93 M solution of phosgene in toluene (1.2 equiv), and
stirred at 0 °C for 2 h. The reaction mixture was extracted twice with
the appropriate aliquots of chilled 0.5M HCl, water, and brine. The
DCM layer was dried over Na2SO4 and treated with a solution of the
appropriate Boc-hydrazine (1-5, 2 equiv) and DIEA (2 equiv) in DCM.
The reaction was stirred at RT for 16 h. The solvent was evaporated,
and the products were purified by silica gel chromatography.
Boc-AzaPhe-L-Ala-OBn (11, 0.75 g, 95% yield): mp ) 108-109
°C; Rf ) 0.84 (10% MeOH in DCM); [R]20 2.3° (c 1.0, CHCl3); H
1
D
NMR 300 MHz (CDCl3): δ 7.40-7.23 (m, 10H), 5.98 and 5.95 (2 s,
N-H’s, 2H), 5.21 (d, 1H, J ) 12 Hz), 5.15 (d, 1H, J ) 12 Hz), 4.59
(q, 1H, J ) 7 Hz), 1.45 and 1.42 (s and d, 12H);13C NMR 75 MHz
(CDCl3): δ 173.6, 157.1, 154.4, 136.2, 135.6, 129.1, 128.9, 128.7,
128.5, 128.2, 127.9, 82.4, 67.1, 50.7, 49.4, 28.4, 19.2; HRMS (FAB)
m/e: 428.2185 (calcd for C23H30N3O5 (M + H+), 428.2179).
General Protocol for the Synthesis of N-Boc-Aza1-Dipeptides
(16-21). N-Boc-aza1-dipeptide benzyl esters (10-15) were dissolved
(62) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal. Biochem.
1970, 34, 595.
(61) DeGrado, W. F.; Kaiser, E. T. J. Org. Chem. 1980, 45, 1295.
9
J. AM. CHEM. SOC. VOL. 126, NO. 21, 2004 6763

A R T I C L E S
Melendez and Lubell
FVPTDVGPFAF-NH2 (25). Purity >99% by LCMS (tR ) 15.1);
LRMS calcd for C60H83N12O14 (M + H+), 1195.6; found, 1195.6.
PTDVGPazaFAF-NH2 (26). Purity 91% by LCMS (tR ) 13.2); LRMS
calcd for C45H64N11O12 (M + H+), 950.5; found, 950.5. PTDVGaza-
PFAF-NH2 (27). Purity 88% by LCMS (tR ) 12.7); LRMS calcd for
C45H64N11O12 (M + H+), 950.5; found, 950.5. azaFVPTDVGPFAF-
NH2 (28). Purity >99% by LC-MS (tR ) 17.0); LRMS calcd for
C59H82N13O14 (M + H+), 1196.6; found, 1196.3. FVPTDVGPazaFAF-
NH2 (29). Purity 92% by LC-MS (tR ) 14.3); LRMS calcd for
C59H82N13O14 (M + H+), 1196.6; found, 1196.6.
Technologies (FQRNT), Valorisation-Recherche Que´be´c (VRQ),
and the Conseil de Recherches en Sciences Naturelles et en
Ge´nie de Canada (CRSNG). We thank Dalbir S. Sekhon for
assistance with LC-MS. R.E.M. thanks the FQRNT for a
postdoctoral fellowship.
Supporting Information Available: Experimental procedures
and spectral data for compounds 1-22 and LC-MS spectra
for 24-29 (PDF). This material is available free of charge via
the Internet at http://pubs.acs.org.
Acknowledgment. This work was supported by grants from
the Fonds Que´be´cois de la Recherche sur la Nature et les
JA039643F
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