Solution-phase submonomer diversification of aza-dipeptide building blocks and their application in aza-peptide and aza-DKP synthesis

Article abstract of DOI:10.1002/psc.1235

Aza-peptides have been used as tools for studying SARs in programs aimed at drug discovery and chemical biology. Protected aza-dipeptides were synthesized by a solution-phase submonomer approach featuring alkylation of N-terminal benzophenone semicarbazon

Full text of DOI:10.1002/psc.1235

Research Article
Received: 22 January 2010
Revised: 9 March 2010
Accepted: 10 March 2010
Published online in Wiley Interscience:
(www.interscience.com) DOI 10.1002/psc.1235
Solution-phase submonomer
diversification of aza-dipeptide
building blocks and their application
in aza-peptide and aza-DKP synthesis
Carine B. Bourguet, Caroline Proulx, Sophie Klocek, David Sabatino
and William D. Lubell∗
Aza-peptides have been used as tools for studying SARs in programs aimed at drug discovery and chemical biology. Protected
aza-dipeptides were synthesized by a solution-phase submonomer approach featuring alkylation of N-terminal benzophenone
semicarbazone aza-Gly-Xaa dipeptides using different alkyl halides in the presence of potassium tert-butoxide as base.
Benzophenone protected aza-dipeptide tert-butyl ester 31c was selectively deprotected at the C-terminal ester or N-terminal
hydrazone to afford, respectively, aza-dipeptide acid and amine building blocks 36c and 40c, which were introduced into
longer aza-peptides. Alternatively, removal of the benzophenone semicarbazone protection from aza-dipeptide methyl esters
29a–c led to intramolecular cyclization to produce aza-DKPs 39a–c. In light of the importance of aza-peptides and DKPs as
therapeutic agents and probes of biological processes, this diversity-oriented solution-phase approach may provide useful
c
tools for studying peptide science. Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article
Keywords: alkylation; aza-Gly-Xaa; aza-peptide; β-turn conformation; benzophenone semicarbazone protecting group; DKP; aza-DKP;
triazine
Introduction
The prerequisite to synthesize the N-alkyl hydrazine prior to
incorporation into aza-peptide has limited the diversity of the aza-
residuesidechain,becauseoftheinherentdifficultytodifferentiate
selectively the two nitrogen of the hydrazine moiety [27]. Alter-
natively, alkylation of a suitably N-protected aza-glycine moiety
surmounts the issues associated with hydrazine synthesis [28,29].
Although N-Fmoc-aza-glycine acid chloride 4 could not be intro-
duced into aza-peptide in solution, likely because of competitive
formation of oxadiazalone 6 (Figure 2) [23], aza-Gly peptides were
synthesized using the activated methylidenecarbazate 20 from
mixing benzophenone hydrazone 17 and p-nitrophenyl chlorofor-
mate 18 (Scheme 2) [28,29]. Subsequent alkylation of solid-phase
bound benzophenone semicarbazone-protected aza-Gly peptide,
semicarbazone deprotection, peptide elongation and cleavage
has provided a submonomer approach for aza-peptide synthesis
[29].Tocomplimentthissolid-phaseapproach,solution-phasesyn-
thesis by a submonomer approach has been explored. At the time,
we were also aware that alkylation of N-phthalimido aza-glycine
tert-butyl ester (N-tert-butyloxycarbonyl-aminophthalimide 13,
Scheme 1) had been achieved by using different alcohols under
Mitsunobu conditions [30]. Both phthalimide and benzophe-
Peptide mimics have been used to enhance the bioavailability,
potency, specificity and duration of action of natural parent
peptides [1–3]. Investigations in peptide mimicry have also shown
that certain structural changes, when introduced into a peptide,
can prevent enzyme degradation and induce conformational
preferences that mimic the biologically active secondary structure
[4]. Among the modifications found in peptide mimicry, aza-
peptides, in which one or more residue is replaced by an
aza-amino acid [5], have found promising utility in enhancing
the drug-like character of peptide candidates [6], by increasing
the duration of action [7] and resistance to enzymatic degradation
[8,9] (see Figure 1 for relevant examples). Furthermore, aza-amino
acid residues have been shown to stabilize β-turn conformations
in peptides as detected by computational [10–12], spectroscopic
[13–15] and crystallographic analyses [13].
The introduction of aza-amino acids into peptides involves
a combination of hydrazine and peptide chemistry (Figure 2)
[5]. For example, hydrazine precursors were used in solution to
synthesize N-Boc protected aza1-dipeptide 3 [20,21] and N-Fmoc
protected aza-amino acid chloride 4, which were used successfully
insolid-phasesynthesestointroduceaza-residuesintobiologically
relevant peptides [22,23]. On the other hand, attempts to prepare
aza-peptide by the reaction of protected N-alkyl hydrazine [24]
11 onto resin-bound active carbamate 9 or isocyanate 10 have
suffered from low yields due to the formation of hydantoin 12
upon intramolecular acylation of the amide of the preceding
C-terminal residue (Figure 2) [24–26].
∗
´
´
Correspondence to: Prof. William D. Lubell, Departement de Chimie, Universite
´
´
´
de Montreal, C.P. 6128. SuccursaleCentre-Ville, Montreal, Quebec, Canada H3C
3J7. E-mail: lubell@chimie.umontreal.ca
´
´
´
Departement de Chimie, Universite de Montreal, C.P. 6128, Succursale
´
´
Centre-Ville, Montreal, Quebec, Canada H3C 3J7
c
J. Pept. Sci. 2010; 16: 284–296
Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd.

SOLUTION-PHASE SUBMONOMER AZA-PEPTIDE AND AZA-DKP SYNTHESIS
R
R
N
N
H
N
H
O
O
Amino-acid
Aza-amino acid
Glp-His-Trp-Ser-Tyr-D-Ser(t-Bu)-Leu-Arg-Pro-aza-Gly-NH2
Goserelin Acetate (Zoladex®)16
used for treatment of prostate and breast cancer
N
N
O
HO
O
N
O
N
AcHN
t-Bu
NH
O
O
NH
MeO
NH
O
N
N
N
O
O
H
H
N
N
O
N
NH2
CO2H
HO
Ph
H
N
H
O
O
O
NH
O
t-Bu
N
H
OMe
HN
Aza-peptide I
Ergotamine containing
AzaPhe analogue19
Atazanavir
inhibitor of HIV-1 protease18
β-sheet breaking peptide for potential
treatment of Alzheimer's disease17
Figure 1. Representive biologically active aza-peptide and aza-DKP analogs [16–19].
aza1-dipeptide
O
R1
N
O
H
N
R3
RO
N
OH
H
N
H
O
R2
H2N
Activation of growing peptide
O
3: R = tBu
2
O
R3
R1
NH
H
N
H
N
O
X
N
H
O
N
H
O
R1
N
O
R3
O
RO
R2
O
O
R3
H
N
H
N
9
H
N
11
X
X
H2N
N
RO
N
H
or
N
H
H
X = leaving
group
R2
O
O
O
R3
R2
O
H
N
1
O C
R3
8
N
N
H
O
R3
R2
O
H
10
H2N
N
R1
N
H
O
O
R2
HN
H
N
R2
O
N
Cl
RO
N
H
N
5
R1 = H
O
O
4: R = Fmoc
O
12
O
O
RO
RO
OH
O
N
N
N
NH
7
6
N- protected aza-amino acid chloride
Figure 2. Methods and pitfalls to avoid in introducing aza-residues into peptides.
none semicarbazone protection strategies were studied to avoid
oxadiazolone formation during incorporation of aza-Gly residues
and to orient regioselective nitrogen alkylation for the synthesis
of protected aza-dipeptide building blocks in solution. Although
N-phthalimido aza-glycinamides could not be successfully diver-
sified, a variety of N-benzophenone semicarbazone aza-glycine
analogs were alkylated to furnish a diverse set of aza-dipeptides.
Moreover, selective removal of the benzophenone or the ester
protection gave suitable building blocks for aza-peptide synthesis,
respectively, by N- or C-terminal elongation. Finally, examination
of the removal of the N-protection from the aza-dipeptide methyl
ester under acidic conditions has provided access to a relatively
rare class of heterocycles, the so-called aza-diketopiperazines
(aza-DKPs). This class of triazines merits study in light of the broad
spectrum of biological activities associated with the parent DKP
structure [31].
Results and Discussion
Synthesis and Attempted Alkylation of Phthalimido Gly-Xaa
Dipeptides
As mentioned earlier, phthalimide protection has been used
for the synthesis of N-alkyl, N-acyl hydrazines in solution- and
on solid-phase [30,32–35]. Mitsunobu alkylation of N-acyla-
minophthalimides provided good yields of alkylated products
using primary, secondary and benzyl alcohols [30]. Removal
of the phthaloyl group with methylhydrazine afforded the
1,1-disubtituted hydrazines [30]. In light of the success with
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J. Pept. Sci. 2010; 16: 284–296 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

BOURGUET ET AL.
Scheme 1. Synthesis and attempted alkylation of phthalimido aza-Gly-Phe-OMe 15.
N-acylaminophthalimides 13, the related ureidophthalimide 15
was synthesized and examined as a protected aza-Gly residue to
explore related alkylation chemistry to provide other aza-amino
acids (Scheme 1).
starting material was obtained from attempts to alkylate N-
ureidophthalimide 15 using allyl bromide or allyl iodide in the
presence of potassium tert-butoxide in THF at 0 ◦C (Scheme 1).
Both the low coupling yields and the lack of alkylation of
ureidophthalimide 15 suggested that an alternative protecting
group strategy was needed to build and modify the aza-Gly-Xaa
dipeptide.
N-aminophthalimide 14 was synthesized as reported [36],
reacted with p-nitrophenyl chloroformate at 0 ◦C for 2 h and
coupled with Phe-OMe in a sluggish reaction giving a mixture
of multiple products, as detected by TLC analysis, from which
silica gel column chromatography finally provided the desired
N-phthalimido aza-Gly-Phe-OMe 15, albeit in 2% yield. Attempts
failed to add diversity onto the phthalimido aza-Gly residue of 15
using benzyl and allyl alcohols under Mitsunobu conditions, which
were reported to be successful for alkylation of Pht-aza-Gly-OtBu
13[30], likelyduetotheweakeracidityofthe N-ureidophthalimide
versus the N-carbamatophthalamide. Furthermore, recovered
Benzophenone Semicarbazone Protection for aza-Gly-Xaa
Synthesis
Hydrazone protection has recently been used to prepare activated
aza-Gly analogs without oxadiazolone formation [28,29]. More-
over, the alkylation of supported aza-Gly peptide has been used
to make aza-peptide on solid phase [29].
Scheme 2. Hydrazone activation and insertion of aza-glycine into dipeptides.
c
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SOLUTION-PHASE SUBMONOMER AZA-PEPTIDE AND AZA-DKP SYNTHESIS
Scheme 3. General method for alkylation of benzophenone protected aza-Gly-Xaa-Y.
Table 1. Alkylated benzophenone protected aza-Gly-Xaa-Y
Entry
Starting material
Y
R1, R2
R3
RX
Z
Product
% Yields
1
24
24
24
24
23
25
25
25
25
21
26
27
27
28
OCH3
OCH3
(CH2)3
(CH2)3
(CH2)3
(CH2)3
H, H
H
H
HC CCH2Br
CH2 CHCH2Br
BnBr
OCH3
OCH3
29a
29b
29c
29d
30
37
20
15
80
14
72
56
84
73
60
51
22a
63b
48
2
3
OCH3
H
OCH3
4
OCH3
H
CH3I
OCH3
5
OCH3
Bn
H
BnBr
OCH3
6
OC(CH3)3
OC(CH3)3
OC(CH3)3
OC(CH3)3
OCH2CH3
OH
(CH2)3
(CH2)3
(CH2)3
(CH2)3
H, H
HC CCH2Br
CH2 CHCH2Br
BnBr
OC(CH3)3
OC(CH3)3
OC(CH3)3
OC(CH3)3
OCH2CH3
OCH2C CH
OCH2C CH
OBn
31a
31b
31c
31d
32
7
H
8
H
9
H
CH3I
10
11
12
13
14
H
CH2 CHCH2Br
HC CCH2Br
HC CCH2Br
BnBr
(CH2)3
H, H
H
33
OH
Bn
Bn
H
34a
34b
35
OH
H, H
NHCH(Ph)2
(CH2)3
HC CCH2Br
NHCH(Ph)2
a Benzophenone aza-Gly-D-Phe-OCH2CH CH was obtained as major side product determined by LCMS analyses.
b Benzophenone aza-Gly-D-Phe-OBn was obtained as major side product determined by LCMS analyses.
In solution, benzophenone semicarbazone-protected aza-Gly
dipeptides21–27weresynthesizedin41–62%yieldsbyactivation
of diphenylhydrazone 17 with p-nitrophenyl chloroformate 18 in
DCM at 0 ◦C for 1 h, followed by addition of a solution of the
coupling partner (amino acid or ester, Xaa-Y as hydrochloride or
free base) and DIEA in DCM or methanol (Scheme 2) [28]. The
reagents CDI [37] and phosgene [22] in solution gave exclusively
symmetric urea 19.
Activated carbazate 20 was coupled to amino methyl ester
hydrochlorides with best conditions involving a premixing of the
salt with 2 eq. of DIEA in DCM prior to addition. Proline tert-
butyl ester was used as the free amine with only 1 eq. of DIEA,
which gave the desired aza-Gly dipeptide 25 in 51% yield. The
amino acids Pro and D-Phe were also coupled to the p-nitrophenyl
carbazate 20 with 1 eq. of DIEA, using methanol as solvent in
the case of proline to furnish benzophenone protected aza-Gly-
Pro 26 and aza-Gly-Phe 27 in 57% and 41% respective yields.
Benzophenone protected aza-Gly-Pro diphenylmethylamide 28
was synthesized in 96% yield as previously described by coupling
the corresponding aza-Gly-Pro 26 and diphenylmethylamine [28].
regioselective alkylation was achieved on methyl ester 24, using
propargyl bromide (37%), benzyl bromide (15%), allyl bromide
(20%) and methyl iodide (80%; Table 1 entries 1–4 and Scheme 3).
Four products were initially obtained from alkylation with
propargyl bromide on benzophenone semicarbazone-protected
aza-Gly-Pro-OMe 24 using potassium tert-butoxide (Figure 3). The
aza-propargylGly-Pro-OMe 29a was isolated in 20% yield and
shown to be contaminated with its corresponding acid and
propargyl ester counterparts 36a and 33, as well as hydantoin 37
in, respectively, 10%, 7% and 20% yields. Intramolecular acylation
of the semicarbazone anion by the methyl ester may likely lead
to the formation of hydantoin 37 which was isolated in 20% yield
and characterized by 1H and 13C NMR.
Residual hydroxide ion caused likely the solvolysis of the methyl
ester, leading to alkylation of the resulting carboxylate to prepare
propargyl ester 33. Alkylation conditions were subsequently
improved to yield the aza-propargylGly-Pro-OMe 29a in 37%
yield (Table 1, entry 1). Ions characteristic of similar side products
weredetectedintheLCMStracesofsemicarbazone-protectedaza-
allylGly-Pro-OMe 29b and aza-Phe-Pro-OMe 29c (Table 1, entries
2 and 3); however, only starting material (20%) and aza-Ala-Pro-
OMe 29d (80%) were recovered from alkylation with iodomethane
(Table 1, entry 4). Attempts to effect alkylation under Mitsunobu
conditions failed. Isomerization of the tertiary amide in aza-Gly-
Pro analogs may be partially responsible for the low yields of
methyl ester 24. A similar attempt to alkylate benzophenone
semicarbazone-protected aza-Gly-D-Phe-OMe 23 using benzyl
bromide in the presence of potassium tert-butoxide (1.1 eq.)
gave aza-Phe-D-Phe-OMe 30 in only 14% yield (Table 1, entry
5). Similarly, aza-allylGly-Gly-OEt 32 was synthesized in 60%
yield by alkylation of benzophenone semicarbazone-protected
aza-Gly-Gly-OEt 21 (Scheme 3 and Table 1, entry 10). Neither
Alkylation of Benzophenone Semicarbazones
Alkylation of the benzophenone imines of glycine esters was first
introduced by O’Donnell as a practical method for the synthesis
of racemic and optically active α-amino acids [38]. In analogy
to the Gly Schiff-base, in which imine double-bond resonance
favorsstabilizationoftheα-carbanion[39],semicarbazones21–28
could be regioselectively deprotonated and alkylated. A general
protocol was explored in which the aza-Gly dipeptide was treated
with potassium tert-butoxide in THF at 0 ◦C under argon followed
by treatment with alkyl halide. According to this procedure, the
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BOURGUET ET AL.
tert-butyl ester was removed by treatment with bubbling HCl in
DCM for 2 h and acids 36a–d were directly used without further
purificationinamidecouplings.Forthesynthesisofamides35a–d,
isobutyl chloroformate and NMM at −15 ◦C proved most effective
giving 12–74% yields (Scheme 4). Other conditions for tert-butyl
ester removal were less successful often causing benzophenone
removal as observed by LCMS analyses [41–43].
O
O
OH
O
OCH3
O
N
N
N
N
N
N
+
36a (10%)
29a (20%)
O
O
N-Terminal deprotection and aza-diketopiperazine formation
O
O
N
N
N
Previously, N-benzophenone semicarbazone aza-Gly-Pro-OMe 24
was converted to hydrochloride salt 38 in 99% yield using 1 N
HCl in THF at 40 ◦C for 12 h [28]. On the other hand, applying the
same conditions to the alkylated methyl esters 29a–c afforded
aza-DKPs 39a–c (Scheme 5).
N
N
+
N
O
37 (20%)
33 (7%)
The synthesis and activity of aza-DKPs has been rather
unexplored [44], yet offers interesting potential for studying
the influence of Cα-stereochemistry on activity. In this light,
the pioneering synthesis of the aza-DKP, c-[aza-Phe-Pro], and its
incorporation into an analog of the peptidic moiety of ergotamine
(Figure 1), led to a notable rearrangement giving imidazolinone
surrogates of the oxazolidin-4-one moiety characteristic of
ergotamine and related ergopeptins [19,45]. Aza-DKPs have been
previously synthesized by condensations of proline esters with
preformed Boc-aza-amino acid chlorides [44] and nitrophenyl
esters [46]. Our entry to aza-DKPs offers efficient means for
diversifying the aza-residue, and may be useful for examining
active DKPs [47,48], and related triazines [49–51].
Figure 3. Aza-propargylGly-Pro-OMe and side products obtained from
alkylation of methyl ester 24.
1
C- nor N-amide alkylation was observed by a H–1H COSY NMR
experiment which indicated a three-bond coupling between the
urea NH and the neighboring αCH2 of the glycine residue.
tert-Butyl protection alleviated the issues of trans-esterification
and low yields in the alkylation of aza-Gly-Pro dipeptide methyl
esters. Under similar alkylation conditions using tert-BuOK and
propargyl, benzyl and allyl bromides, semicarbazone-protected
aza-propargylGly-Pro, aza-allylGly-Pro and aza-Phe-Pro tert-butyl
esters 31a–c were afforded in improved yields (56–84%); methyl
iodidegaveaza-Ala-Prodipeptidetert-butylester31din73%yield.
Alkylation of aza-Gly-dipeptide acids was also achieved by dou-
ble deprotonation with 2 eq. of base and treatment with excess
alkyl halide to provide the N,O-dialkylated products. Benzophe-
none semicarbazone-protected aza-Gly-Pro 26 and aza-Gly-D-Phe
27 reacted with propargyl bromide under these conditions
to give the aza-propargylGly-Pro and aza-propargylGly-D-Phe
propargyl esters 33 and 34a in 51 and 22% yields, respectively
(Table 1, entries 11 and 12) [40]. Treatment of benzophenone
semicarbazone-protected aza-Gly-D-Phe 27 with benzyl bromide
gave aza-Phe-D-Phe benzyl ester 34b in 63% yield (Table 1,
entry 13).
N-terminal deprotection without aza-DKP formation
and enantiomeric purity
To avoid aza-DKP formation and afford a free amine building
block for aza-peptide synthesis, N-benzophenone semicarbazone
aza-Phe-Pro tert-butyl ester 31c was treated with hydroxylamine
hydrochloride in pyridine at 60 ◦C for 12 h [29]. Purification by
chromatography on silica gel gave the free amine 40c in 88% yield
(Scheme 6).
Amine 40c was subsequently used for determining enan-
tiomeric purity to ascertain whether α-carbon racemization had
occurred during semicarbazone alkylation and removal. Aza-
tripeptides (R,S)- and (S,S)-41 were synthesized by coupling 40c,
respectively, to Fmoc-D- and L-alanine by the way of a mixed
anhydride, formed using isobutylchloroformate and NMM. With-
out purification, aza-tripeptide (S,S)-41 was then demonstrated
to be of >98% diastereomeric excess using normal phase HPLC
with subsequent incremental additions of the (R,S)-41 diastere-
omer to establish the limits of detection (Scheme 6; for HPLC
analyses see supporting information). Hence, no racemization was
deemed to occur during the submonomer aza-peptide synthe-
sis leading to pure (S,S)-41 and (R,S)-41 diastereomers. Moreover,
the use of the N-benzophenone semicarbazone aza-dipeptide
tert-butyl ester as building block was validated by the synthe-
sis of these aza-tripeptides and their purification in 92 and 95%
yields.
Finally, alkylation of aza-Gly-Pro dipeptide amide 28 with
propargyl bromide and potassium tert-butoxide provided aza-
propargylGly-Pro amide 35 in 48% yield (Table 1, entry 14).
Examination of the 1H-1H COSY spectra of 35 showed the coupling
between the amide and the methyl protons of the diphenylmethyl
amide indicating that no N-alkylation occurred at the C-terminal
amide.
C-Terminal deprotection and coupling of N-benzophenone
aza-dipeptides
N-Protected aza1-dipeptides have been used as configurationally
stable building blocks for assembling aza-peptides in solution
and on solid phase for subsequent SAR studies [21,26]. With
means in hand for alkylation of N-benzophenone semicarbazone
aza-Gly-Xaa dipeptide esters to generate diverse aza-dipeptide
analogs, ester removal was next examined to provide the building
blocks for introduction into longer peptides. Methyl esters 29a–d
were hydrolyzed using LiOH (2 eq.) in 3 : 1 methanol : water to
give acids 36a–d in 38–89% yield. Acid 36a was also prepared
in 72% yield by the treatment of tert-butyl ester 31a with 8 : 2
TFA in DCM for 12 h followed by chromatography. Alternatively,
Conclusion
The construction and alkylation of aza-Gly dipeptides to prepare a
variety of aza-dipeptides in solution has been achieved using
benzophenone semicarbazone protection. Using a variety of
alkyl halides, methyl, allyl and propargyl side chains, which
c
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SOLUTION-PHASE SUBMONOMER AZA-PEPTIDE AND AZA-DKP SYNTHESIS
Scheme 4. C-terminal deprotection of benzophenone semicarbazone aza-dipeptide esters.
for preparing diverse analogs of these interesting structures in
solution.
Experimental
Unless otherwise noted, all reactions were performed under an
argon atmosphere and distilled solvents were transferred by sy-
ringe. Benzophenonehydrazoneandp-nitrophenylchloroformate
were purchased from Aldrich Chemicals. Anhydrous solvents (THF,
CH2Cl2 and CH3OH) were obtained by passage through solvent
filtration systems (GlassContour, Irvine, CA, USA). DIEA was dis-
tilled over ninhydrin and CaH2. Final reaction mixture solutions
were dried over MgSO4 or Na2SO4. Chromatography was on
230–400 mesh silica gel, and TLC was on glass-backed silica
plates. Melting points were made on a Gallankamp apparatus
and are uncorrected. Specific rotations [α]D were measured at
20 ◦C at the specified concentrations (c in g/100 ml) using a
1-dm cell on a PerkinElmer Polarimeter 341 and the general for-
Scheme 5. Deprotection of benzophenone semicarbazone and aza-DKP
synthesis.
20
mula: [α]D = (100 × α)/(d × c). Accurate mass measurements
were performed on a LC-MSD instrument from Agilent techno-
logies in positive electrospray (ES) mode at the Univer-
site´ de Montre´al Mass Spectrometry facility. Analytical HPLC
for enantiomeric purity determination was made by using
an analytical Phenomenex Luna normal phase HPLC column
(150 × 4.60 mm, 3 µm). Either protonated molecular ions [M
+ H]+ or sodium adducts [M + Na]+ were used for em-
pirical formula confirmation. 1H NMR spectra were measured
in CDCl3, CD3OD or DMSO-d6 at 400 MHz and referenced to
CDCl3 (7.26 ppm), CD3OD (3.31 ppm) or DMSO-d6 (2.50 ppm).
13C NMR spectra were measured in CDCl3, CD3OD or DMSO-d6
at 100 MHz and, respectively, referenced to CDCl3 (77.0 ppm),
CD3OD (49.0 ppm) or DMSO (39.52 ppm). Coupling constants,
J values were measured in hertz (Hz) and chemical shift values in
parts per million (ppm).
Scheme 6. Synthesisanddiastereomericpurityofaza-tripeptides(R,S)-and
(S,S)-41.
Synthesis of Phtalimido Aza-Glycinyl-Phenylalanine-OMe (15)
are challenging to introduce by traditional hydrazine chemistry,
were incorporated specifically onto the aza-Gly residue by
this submonomer synthesis. C-terminal amino acid residues
bearing aliphatic and aromatic side chains were tolerated such
that removal of the ester and benzophenone semicarbazone
protection could be accomplished selectively to afford dipeptide
units for elongation on the C- and N-termini, respectively, to
afford aza-peptides with diastereomeric purity >98%. Using
acidic conditions to remove the benzophenone semicarbazone
moiety from aza-Xaa-Pro methyl esters gave aza-DKPs 39a–c
possessing a diverse series of side chains at the aza-residue. By
circumventing the issues of hydrazine synthesis, this method
for aza-dipeptide and aza-DKP synthesis has opened the way
N-Aminophthalimide 14 (2 g, 12.3 mmol, prepared according to
the literature procedure [36]) was added to a solution of p-
nitrophenylchloroformate (2.5 g, 12.3 mmol) in anhydrous DCM
(31 ml) at 0 ◦C under argon. The reaction mixture was stirred at
room temperature for 2 h, and when no more starting material
appeared by TLC (Rf = 0.5, 1 : 1 v/v hexanes : EtOAc), the reaction
was treated dropwise with DIEA (2.1 ml, 12.3 mmol), stirred for
15 min and treated with a premixed solution of Phe-OMe•HCl
(2.7 g, 12.4 mmol) and DIEA (2.1 ml, 12.3 mmol) in CH2Cl2. After
stirring at room temperature for 12 h, the mixture was diluted with
CH2Cl2 (100 ml)andextractedwithasaturatedsolutionofaqueous
NaHCO3 (3×100 ml)andbrine(1×100 ml). Thecombinedorganic
c
J. Pept. Sci. 2010; 16: 284–296 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

BOURGUET ET AL.
phases were dried over MgSO4 and evaporated under reduced
pressure. The crude product was purified by flash chromatography
on silica gel with a 1 : 1 mixture of hexanes : ethyl acetate.
Evaporation of the collected fractions gave aza-dipeptide 15 as a
white solid (92 mg, 2%): mp 184–185 ◦C; Rf 0.42 (hexanes : EtOAc
1 : 1); [α]20D 104 (c 0.1, CHCl3); 1H NMR (CDCl3, 400 MHz): δ 3.09 (2H,
m), 3.67 (3H, s), 4.79 (1H, dd, J = 5.8, 2 Hz), 6.06 (1H, d, J = 7.8 Hz),
7.11 (3H, t, J = 7.2 Hz), 7.19 (2H, t, J = 7,2 Hz), 7.51 (1H, s), 7.75 (2H,
dd, J = 5.4, 3.1 Hz), 7.86 (2H, dd, J = 5.4, 3.1 Hz); 13C NMR (CDCl3,
100 MHz): δ 37.6, 52.1, 53.8, 123.5, 126.6, 128.0, 129.1, 129.7, 134.2,
135.3, 154.8, 165.7, 172.3. HRMS (ESI) m/z calcd for C19H18N3O5
368.1249, found [M + H]+ 368.1241.
0.68, CHCl3); 1H NMR (CDCl3, 400 MHz): δ 3.20 (2H, m), 3.73 (3H, s),
4.85 (1H, m), 6.78 (1H, dd, J = 12, 24 Hz), 7.19–7.51 (15H, m) 7.58
(1H, s); 13C NMR (CDCl3, 100 MHz): δ 38.2, 38.4, 52.1, 52.2, 53.3, 53.8,
126.8, 127.1, 128.1, 128.3, 128.4, 128.6, 129.2, 129.3, 129.6, 129.7,
131.5, 135.9, 136, 136.7, 148.2, 154.6, 172.2, 172.9. HRMS (ESI) m/z
calcd for C24H24N3O3 402.1812; found [M + H]+ 402.1825.
Benzhydrylidene Aza-Glycinyl-Proline Methyl Ester (24)
This was synthesized according to the procedure described above
from proline methyl ester hydrochloride (640 mg, 4.96 mmol) and
purified by column chromatography using 1 : 1 hexanes : EtOAc as
eluant. Evaporation of the collected fractions gave semicarbazone
24, as a light yellow oil (1 g, 60%): Rf 0.36 (hexanes : EtOAc 1 : 1);
[α]20D −41.0 (c 1.0, CHCl3). 1H NMR (CDCl3, 400 MHz): δ 1.88–2.06
(3H, m), 2.14–2.23 (1H, m), 3.55–3.65 (2H, m), 3.67 (3H, s), 4.64 (1H,
d, J = 8 Hz), 7.25–7.34 (5H, m), 7.48–7.56 (5H, m), 7.76 (1H, s); 13C
NMR (CDCl3, 100 MHz): δ 24.4, 30.2, 48.2, 52.6, 60.8, 116.4, 126.5,
127.8, 128.6, 128.9, 129.6, 130.2, 132.5, 137.4, 150.5, 154.3, 164.4,
173.8. HRMS (ESI) m/z calcd for C20H22O3N3 [M + H]+ 352.1656;
found 352.1671.
Benzhydrylidene Aza-Glycinyl-Glycine Ethyl Ester (21)
A stirred solution of p-nitrophenyl chloroformate (464 mg,
2.3 mmol) in 20 ml of DCM at 0 ◦C was treated dropwise with
a solution of benzophenone hydrazone 17 (451.4 mg, 2.3 mmol)
in 10 ml of DCM. The ice bath was removed and the reaction
was allowed to warm to room temperature. After 1 h, complete
disappearanceofthehydrazoneandtheformationoftheactivated
methylidine carbazate intermediate 20 were observed by TLC
Rf 0.83 (EtOAc : hexane 1 : 1). The reaction mixture was treated
with a premixed solution of ethyl glycinate hydrochloride (0.32 g,
2.3 mmol) and DIEA (0.8 ml, 4.6 mmol) in DCM. After stirring
overnight, the crude reaction mixture was concentrated under
vacuum and purified by column chromatography on silica gel
using 2 : 1 hexanes : EtOAc as eluant. Evaporation of the collected
fractions gave semicarbazone 21 as a white solid (0.5 g, 60%).
Rf 0.25 (hexanes : EtOAc 2 : 1); mp 140–142 ◦C; 1H NMR (CDCl3,
400 MHz): δ 1.19 (3H, t, J = 7 Hz), 4.02 (2H, d, J = 5.6 Hz), 4.13 (2H,
dd, J = 7, 14.4 Hz), 6.78 (1H, t, J = 5.5 Hz), 7.1–7.4 (10H, m), 7.61
(1H, s); 13C NMR (CDCl3, 100 MHz): δ 13.8, 41.4, 61.0, 126.8, 127.8,
127.9, 128.1, 129.0, 129.1, 129.4, 129.4, 129.5, 131.4, 136.5, 148.2,
151.0, 154.9, 170. HRMS (ESI) m/z calcd for C18H20N3O3 [M + H]+
326.1499; found 326.1503.
Benzhydrylidene Aza-Glycinyl-Proline tert-Butyl Ester (25)
Thiswassynthesizedaccordingtothegeneralproceduredescribed
above from a premixed solution of free amino proline tert-butyl
ester (2.3 g, 11.68 mmol) with 1 eq. of DIEA (2 ml, 11.68 mmol)
in DCM and purified by column chromatography using 3 : 2
hexanes : EtOAc as eluant. Evaporation of the collected fractions
gave semicarbazone 25, as a light yellow foam (2.32 g, 51%): Rf
0.37 (hexanes : EtOAc 1 : 1); [α]20D −42.3 (c 0.208, CHCl3). 1H NMR
(CDCl3, 400 MHz): δ 1.26 (9H, s), 1.73–1.87 (3H, m), 2.06 (1H, s), 3.46
(2H, m), 4.51 (1H, s), 7.08–7.16 (5H, m), 7.38–7.43 (5H, m), 7.68 (1H,
s); 13C NMR (CDCl3, 100 MHz): δ 24.8, 28.2, 31.0, 48.1, 61.0, 81.4,
127.6, 128.5, 128.8, 129.3, 130.0, 130.1, 132.3, 137.6, 148.9, 154.0,
172.1. HRMS (ESI) m/z calcd for C23H28O3N3 [M + H]+ 394.2125;
found 394.2142.
Benzhydrylidene Aza-Glycinyl-Leucine Methyl
Ester (22)
Benzhydrylidene Aza-Glycinyl-Proline (26)
This was synthesized according to the procedure described above
for the synthesis of aza-Gly dipeptide 21 from methyl leucinate
hydrochloride (0.3 g, 1.65 mmol), and the product was purified
by column chromatography using 2 : 1 hexanes : EtOAc as eluant.
Evaporation of the collected fractions gave semicarbazone 22
as a white solid (0.4 g, 62%): Rf 0.47 (2 : 1 hexanes : EtOAc); mp
148–150 ◦C; [α]20D 26.7 (c 0.25, CHCl3). 1H NMR (CDCl3, 400 MHz):
δ 0.91 (6H, dd, J = 4, 8 Hz), 1.55–1.75 (3H, m), 3.67 (3H, s), 4.55 (1H,
dt, J = 3.5, 9.7 Hz), 6.61 (1H, d, J = 8.8 Hz), 7.15–7.46 (10H, m),
7.56 (1H, s); 13C NMR (CDCl3, 100 MHz): δ 13.8, 21.6, 22.6, 24.5, 41.6,
50.9, 51.8, 53.1, 126.8, 128.0, 128.1, 129.0, 129.3, 129.4, 129.5, 131.5,
136.6, 148.1, 154.9, 173.5. HRMS (ESI) m/z calcd for C21H26N3O3
[M + H]+ 368.1969; found 368.1968.
This was synthesized according to the procedure described
above from proline (570 mg, 4.96 mmol) dissolved in methanol
with 1 eq. of DIEA (0.86 ml, 4.96 mmol) and purified by column
chromatography using 20 : 1 EtOAc : AcOH as eluant. Evaporation
of the collected fractions gave semicarbazone 26 as a pale yellow
foam (1 g, 57%): Rf 0.5 (EtOAc : AcOH 20 : 1); mp 86.2–88.2 ◦C;
20
[α]D −97.3 (c 1.09, CHCl3). 1H NMR (CDCl3, 400 MHz): δ 1.94–2.04
(3H, m), 2.28 (1H, s), 3.44–3.51 (2H, m), 4.57 (1H, dd, J = 2.8,
4.8 Hz), 7.28–7.33 (5H, m), 7.50–7.58 (5H, m), 7.92 (1H, br s); 13C
NMR (CDCl3, 100 MHz): δ 24.3, 28.2, 47.3, 60.4, 127.3, 128.1, 128.2,
129.3, 129.7, 129.8, 131.6, 136.8, 150.9, 155.0. HRMS (ESI) m/z calcd
for C19H20O3N3 [M + H]+ 338.1499; found 338.1499.
Benzhydrylidene Aza-Glycinyl-D-Phenylalanine (27)
Benzhydrylidene Aza-Glycinyl-D-Phenylalanine Methyl Ester
(23)
This was synthesized from D-phenylalanine (4 g, 20.4 mmol) in
DCM according to the procedure described above and purified
by column chromatography using 9 : 1 DCM : MeOH as eluant.
Evaporation of the collected fractions gave semicarbazone 27
as a white solid (3.23 g, 41%): Rf 0.35 (MeOH : DCM 1 : 9); mp
This was synthesized from D-phenylalanine methyl ester hy-
drochloride (4 g, 22.3 mmol) according to the procedure de-
scribed above and purified by column chromatography using
1 : 1 hexanes : EtOAc as eluant. Evaporation of the collected frac-
tions gave semicarbazone 23 as a white solid (3.6 g, 44%). Rf 0.32
= 210.1–213.0; [α]20 21.2 (c 0.84, CHCl3). 1H NMR (DMSO-d6,
D
(ether : petroleum ether 4 : 1); mp 119.4–122.6, [α]20 −34.6 (c
400 MHz): δ 3.12–3.17 (2H, m), 4.48 (1H, dd, J = 6.6, 13.6 Hz), 7.09
D
c
www.interscience.com/journal/psc Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2010; 16: 284–296

SOLUTION-PHASE SUBMONOMER AZA-PEPTIDE AND AZA-DKP SYNTHESIS
(1H, d, J = 8.2 Hz), 7.25–7.42 (12H, m), 7.52–7.60 (3H, m), 8.50 (1H,
br s), 12.85 (1H, br s); 13C NMR (DMSO-d6, 100 MHz): δ 38.2, 40.5,
40.8, 41.1, 41.3, 41.6, 54.8, 127.9, 128.0, 129.6, 129.7, 130.3, 130.5,
130.7, 130.8, 133.4, 138.6, 138.7, 148.5, 155.7, 174.6. HRMS (ESI)
m/z calcd for C23H22O3N3 [M + H]+ 388.1656; found 388.1662.
Benzhydrylidene Aza-Alaninyl-Proline Methyl Ester (29d)
This was synthesized according to the procedure described for
29a but using methyl iodide (0.15 ml, 1.25 mmol) as alkylating
agent. After purification by chromatography on silica gel using
20% EtOAc in hexanes as solvent system, the evaporation of
the collected fractions gave the methyl semicarbazone 29d as a
light yellow foam (248 mg, 80%): Rf 0.39 (hexanes : EtOAc 1 : 1);
[α]20D −173 (c 0.33, CHCl3). 1H NMR (CDCl3, 400 MHz): δ 1.84–1.96
(3H, m), 2.13–2.16 (1H, m), 2.76 (3H, s), 3.39 (3H, s), 3.73 (2H, t,
J = 6.8 Hz), 4.56 (1H, dd, J = 3.6 Hz, 8.4 Hz), 7.33–7.39 (5H, m),
7.44–7.48 (5H, m); 13C NMR (CDCl3, 100 MHz): δ 23.7, 30.1, 37.6,
49.4, 51.4, 61.1, 127.8, 127.9, 128.1, 128.5, 128.9, 129.1, 129.5, 136.4,
138.7, 156.7, 159.9, 173.5. HRMS (ESI) m/z calcd for C21H24O3N3 [M
+ H]+ 366.1739; found 366.1814.
Benzhydrylidene Aza-Propargylglycinyl-Proline Methyl Ester
(29a)
This was synthesized from benzophenone aza-Gly-Pro-OMe 24
(400 mg, 1.14 mmol) dissolved in 5 ml of anhydrous THF at 0 ◦C.
Thissolutionwastreatedwith1.1eq.oftBuOK(141 mg,1.25 mmol)
and stirred for 15 min prior to the dropwise addition of 1.1 eq. of
propargyl bromide (112 µl, 1.25 mmol). The reaction was allowed
to warm to room temperature and left to react overnight. After
evaporation under reduced pressure, the crude material was
purified by chromatography on silica gel using 20% EtOAc in
hexanes as solvent system. Evaporation of the collected fractions
gave the propargyl semicarbazone 29a as a pale yellow foam
Benzhydrylidene Aza-Phenylalanilyl-D-Phenylalanine Methyl
Ester (30)
This was synthesized according to the procedure described for 29c
from23 (0.46 mmol) using benzyl bromide (65.8 µl, 0.55 mmol) as
alkylating agent. After purification by chromatography on silica
gelusing 15%EtOAcinhexanesassolventsystem, theevaporation
of the collected fractions gave the benzyl semicarbazone 30 as a
yellow oil (33.3 mg, 14%): Rf 0.52 (1 : 1 hexane : EtOAc); [α]20D 27.6
(c 0.47, CHCl3). 1H NMR (CDCl3, 400 MHz): δ 3.17–3.30 (2H, m), 3.78
(3H, s), 4.45 (1H, d, J = 16.4 Hz), 4.65 (1H, d, J = 16.4 Hz), 4.94 (1H,
dd, J = 7.2 Hz, 13.2 Hz), 6.79 (2H, d, J = 7.2 Hz), 7.08–7.49 (19H,
m); 13C NMR (CDCl3, 100 MHz) δ 38.1, 49.0, 52.1, 54.4, 126.6, 126.9,
127.0, 127.9, 128.3, 128.4, 128.5, 129.1, 129.2, 129.6, 129.7, 135.5,
136.2, 136.5, 138.7, 157.1, 158.3, 172.6. HRMS (ESI) m/z calcd for
C31H29O3N3 [M + Na]+ 514.2101; found 514.2119.
(165 mg, 37%): Rf 0.52 (hexanes : EtOAc 1 : 1); [α]20 −126 (c 0.21,
D
CHCl3). 1H NMR (CDCl3, 400 MHz): δ 1.76–1.83 (1H, m), 1.86–1.94
(2H, m), 2.09 (1H, t, J = 2.4 Hz), 2.10–2.12 (1H, m), 3.45 (3H, s),
3.69–3.76 (3H, m), 4.31 (1H, dd, J = 2.4, 17.6 Hz), 4.52 (1H, dd,
J = 3.2, 8.4 Hz), 7.36–7.38 (2H, m), 7.41–7.43 (3H, m), 7.46–7.48
(3H, m), 7.52–7.56 (2H, m); 13C NMR (CDCl3, 100 MHz): δ 38.1,
49.7, 52.1, 61.3, 72.0, 77.1, 79.6, 128.5, 129.1, 129.2, 129.3, 129.9,
130.5, 136.3, 136.7, 159.9, 161.0, 173.8. HRMS (ESI) m/z calcd for
C23H24O3N3 [M + H]+ 390.1739; found 390.1812.
Benzhydrylidene Aza-Allylglycinyl-Proline Methyl Ester (29b)
Thiswassynthesizedaccordingtotheproceduredescribedfor29a
but using allyl bromide (0.11 ml, 1.25 mmol) as alkyl agent. After
purification by chromatography on silica gel using 20% EtOAc
in hexanes as solvent system, the evaporation of the collected
fractions gave the allyl semicarbazone 29b as a pale yellow foam
Benzhydrylidene Aza-Propargylglycinyl-Proline tert-Butyl
Ester (31a)
This was synthesized from benzophenone aza-Gly-Pro-OtBu 25
(200 mg, 0.51 mmol) dissolved in 2 ml of anhydrous THF at
0 ◦C. This solution was treated with 1.1 eq. of tBuOK (63 mg,
0.56 mmol) and the reaction mixture was stirred for 15 min
followed by dropwise addition of 1.1 eq. of propargyl bromide
(50 µl, 0.56 mmol). The reaction was allowed to warm to room
temperature and left to react overnight. After evaporation
under reduced pressure, the crude material was purified by
chromatography on silica gel using 20% EtOAc in hexanes as
solvent system. Evaporation of the collected fractions gave the
propargyl semicarbazone 31a as a white foam (158 mg, 72%): Rf
(87 mg, 20%): Rf 0.50 (hexanes : EtOAc 1 : 1); [α]20 −179 (c 0.267,
D
CHCl3). 1H NMR (CDCl3, 400 MHz): δ 1.78–2.04 (3H, m), 2.12–2.19
(1H, m), 3.43 (3H, s), 3.63–3.69 (3H, m), 4.11 (1H, dd, J = 5.2 Hz,
16 Hz), 4.50 (1H, dd, J = 3.6 Hz, 8.4 Hz), 4.88 (1H, dd, J = 1.2 Hz,
17.2 Hz), 5.01 (1H, dd, J = 1.2 Hz, 9.2 Hz), 5.61–5.69 (1H, m),
7.34–7.41 (5H, m), 7.46–7.49 (5H, m); 13C NMR (CDCl3, 100 MHz)
δ 23.8, 29.9, 49.1, 51.1, 51.4, 60.8, 116.9, 127.8, 128.5, 128.6, 128.7,
129.2, 129.7, 133.5, 136.4, 138.5, 159.3, 160.1, 173.3. HRMS (ESI)
m/z calcd for C23H26O3N3 [M + H]+ 390.1896; found 390.1969.
0.40 (hexanes : EtOAc 7 : 3); [α]20 −17.6 (c 0.193, CHCl3). 1H NMR
D
Benzhydrylidene Aza-Phenylalaninyl-Proline Methyl Ester
(29c)
(CDCl3, 400 MHz): δ 1.44 (9H, s), 1.74–1.77 (1H, m), 1.86–1.91 (2H,
m), 2.10–2.13 (2H, m), 3.60–3.64 (2H, m), 3.65 (1H, d, J = 2.4 Hz),
3.92 (1H, d, J = 17.6 Hz) 4.49 (1H, m), 7.33–7.36 (2H, m), 7.39–7.45
(6H, m), 7.57–7.59 (2H, m); 13C NMR (CDCl3, 100 MHz): δ 24.6, 28.4,
30.1, 38.7, 49.7, 61.8, 71.9, 79.9, 81.3, 128.5 128.9, 129, 129.2, 129.7,
130.4, 136.4, 138.7, 159.7, 161,1, 172.6. HRMS (ESI) m/z calcd for
C26H30O3N3 [M + H]+ 432.2282; found 432.2301.
This was synthesized according to the procedure described for
29a but using benzyl bromide (0.15 ml, 1.25 mmol) as alkylating
agent. After purification by chromatography on silica gel using
20% EtOAc in hexanes as solvent system, the evaporation of the
collected fractions gave the benzyl semicarbazone 29c as a yellow
foam (77 mg, 15%): Rf 0.53 (hexanes : EtOAc 1 : 1); [α]20 −101
D
1
(c 0.467, CHCl3). H NMR (CDCl3, 400 MHz): δ 1.81–1.95 (3H, m),
Benzhydrylidene Aza-Allylglycinyl-Proline tert-Butyl Ester
(31b)
2.14–2.19 (1H, m), 3.47 (3H, s), 3.68–3.79 (2H, m), 4.36 (1H, d,
J = 16 Hz), 4.55 (1H, dd, J = 3.6 Hz, 8 Hz), 4.76 (1H, d, J = 16 Hz),
7.06–7.45 (15H, m); 13C NMR (CDCl3, 100 MHz): δ 23.9, 29.5, 29.9,
49.3, 51.5, 51.9, 60.9, 126.5, 127.5, 127.7, 127.8, 128.3, 128.6, 128.8,
129.3, 129.6, 136.1, 137, 138.6, 159.3, 160.0, 173.4. HRMS (ESI) m/z
calcd for C27H28O3N3 [M + H]+ 442.2052; found 442.2124.
This was synthesized according to the procedure described for the
synthesis of aza-propargylglycinyl dipeptide 31a but using allyl
bromide (47 µl, 0.56 mmol) as alkylating agent. After purification
by chromatography on silica gel using 20% EtOAc in hexanes as
c
J. Pept. Sci. 2010; 16: 284–296 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

BOURGUET ET AL.
solvent system, the evaporation of the collected fractions gave
Benzhydrylidene Aza-Propargylglycinyl-Proline Propargyl
Ester (33)
the allyl semicarbazone 31b as a yellow oil (124 mg, 56%): Rf
1
0.46 (hexanes : EtOAc 7 : 3); [α]20 −3.6 (c 0.165, CHCl3). H NMR
D
This was synthesized from benzophenone aza-Gly-Pro-OH 26
(200 mg, 0.59 mmol) dissolved in 5 ml of anhydrous THF at
0 ◦C. This solution was treated with 2 eq. of tBuOK (133 mg,
1.18 mmol) and the reaction mixture was stirred for 15 min
followed by the dropwise addition of 5 eq. of propargyl bromide
(0.26 ml, 2.97 mmol). The reaction was allowed to warm to
room temperature and left to react overnight. After evaporation
under reduced pressure, the crude material was purified by flash
chromatography on silica gel using 20% EtOAc in hexanes as
solvent system. Evaporation of the collected fractions gave the
propargyl semicarbazone 33 as a light yellow foam (125 mg, 51%):
(CDCl3, 400 MHz): δ 1.45 (9H, s), 1.65–1.72 (1H, m), 1.84–1.90 (2H,
m), 2.07–2.14 (1H, m), 3.55–3.62 (2H, m), 3.82 (1H, dd, J = 6 Hz,
15.9 Hz), 4.02 (1H, dd, J = 5.6 Hz, 15.9 Hz), 4.39 (1H, dd, J = 4.4 Hz,
8.4 Hz), 4.93 (1H, dd, J = 1.6 Hz, 17.2 Hz), 5.02 (1H, dd, J = 1.2 Hz,
10.4 Hz), 5.64–5.71 (1H, m), 7.32–7.45 (8H, m), 7.52–7.55 (2H,
m); 13C NMR (CDCl3, 100 MHz): δ 24.8, 28.4, 30.0, 49.7, 52.2, 61.8,
81.1, 117.3, 128.4, 128.9, 129.2, 129.5, 130.1, 134.4, 137.0, 139.0,
159.7, 160.4, 172.7. HRMS (ESI) m/z calcd for C26H32O3N3 [M + H]+
434.2607; found 434.2481.
Rf 0.42 (hexane : EtOAc 1 : 1); [α]20 −225 (c 0.09, CHCl3). 1H NMR
Benzhydrylidene Aza-Phenylalaninyl-Proline tert-Butyl Ester
(31c)
D
(CDCl3, 400 MHz): δ 1.83 (1H, m), 1.92–1.99 (2H, m), 2.03 (1H, s),
2.08–2.16 (1H, m), 2.41 (1H, s), 3.62–3.72 (3H, m), 4.32 (1H, d,
J = 2 Hz), 4.36 (1H, d, J = 2 Hz), 4,52 (2H, m), 7.31–7.51 (10H, m);
13C NMR (CDCl3, 100 MHz): δ 23.2, 29.3, 29.7, 37.1, 49.0, 51.5, 60.6,
71.4, 74.6, 76.5, 77.2, 78.7, 127.8, 127.9, 128.3, 128.4, 128.5, 129.3,
129.8, 135.5, 138.0, 159.3, 160.1, 171.5. HRMS (ESI) m/z calcd for
C25H24O3N3 [M + H]+ 414.1812; found 414.1826.
This was synthesized according to the procedure described for
31a but using benzyl bromide (67 µl, 0.56 mmol) as alkylating
agent. After purification by chromatography on silica gel using
20% EtOAc in hexanes as solvent system, the evaporation of the
collected fractions gave the benzyl semicarbazone 31c as a yellow
oil (207 mg, 84%): Rf 0.52 (hexanes : EtOAc 7 : 3); [α]20 10 (c 0.22,
D
CHCl3). 1H NMR (CDCl3, 400 MHz): δ 1.49 (9H, s), 1.71–1.78 (1H, m),
1.82–1.98 (2H, m), 2.14–2.19 (1H, m), 3.59–3.68 (2H, m), 4.42–4.46
(1H, m), 4.58 (1H, d, J = 16 Hz), 4.66 (1H, d, J = 16 Hz), 7.12–7.18
(3H, m), 7.22–7.29 (6H, m), 7.42–7.43 (6H, m); 13C NMR (CDCl3,
100 MHz): δ 24.9, 28.4, 30.1, 49.9, 53.2, 62.0, 81.2, 127.0, 128.3,
128.4, 128.6, 128.8, 128.9, 129.3, 129.6, 130.1, 136.8, 137.9, 139.1,
159.6, 160.4, 172.7. HRMS (ESI) m/z calcd for C30H34O3N3 [M + H]+
484.2414; found 484.2641.
Benzhydrylidene Aza-Propargylglycinyl-D-Phenylalanine
Propargyl Ester (34a)
This was synthesized from benzophenone aza-Gly-D-Phe-OH
27 (500 mg, 1.29 mmol) according to the procedure described
for dialkylated aza-analog 33 using propargyl bromide. After
purification by flash chromatography using a gradient solvent of
1 : 9 ethyl acetate in hexanes, the evaporation of the collected
fractions afforded 34a as a yellow oil in 22% yield (131 mg). Rf 0.61
1
(hexanes : EtOAc 1 : 1); [α]20 50.5 (c 1.0, CHCl3). H NMR (CDCl3,
Benzhydrylidene Aza-Alaninyl-Proline tert-Butyl Ester (31d)
D
400 MHz): δ 2.06 (1H, s), 2.51 (1H, s), 3.21–3.24 (2H, m), 3.85 (1H, d,
J = 20.4 Hz), 4.19 (1H, d, J = 18 Hz), 4.24 (1H, d, J = 2.4 Hz), 4.78
(1H, d, J = 2.4 Hz), 4.89 (1H, m), 7.01 (1H, d, J = 7.7 Hz), 7.21–7.28
(5H, m), 7.32–7.41 (4H, m), 7.44–7.46 (6H, m); 13C NMR (CDCl3,
100 MHz): δ 35.0, 37.8, 52.4, 54.2, 71.8, 75.2, 78.3, 126.9, 128.0,
128.5, 129.0, 129.4, 129.6, 130.0, 135.2, 135.7, 138.2, 157.6, 158.2,
171.0. HRMS (ESI) m/z calcd for C29H26O3N3 [M + H]+ 464.1968;
found 464.1991.
This was synthesized according to the procedure described for
31a but using methyl iodide (35 µl, 0.56 mmol) as alkylating
agent. After purification by chromatography on silica gel using
20% EtOAc in hexanes as solvent system, the evaporation of
the collected fractions gave the methyl semicarbazone 31d as
a light yellow oil (152 mg, 73%): Rf 0.38 (hexanes : EtOAc 7 : 3);
[α]20 −9.7 (c 0.27, CHCl3). 1H NMR (CDCl3, 400 MHz): δ 1.43 (9H,
D
s), 1.72–1.76 (1H, m), 1.77–1.95 (2H, m), 2.14–2.19 (1H, m), 2.77
(3H, s), 3.63–3.74 (2H, m), 4.51 (1H, dd, J = 4.4 Hz, 8 Hz), 7.29–7.35
(5H, m), 7.41–7.43 (3H, m), 7.52–7.54 (2H, m); 13C NMR (CDCl3,
100 MHz): δ 24.7, 28.4, 30.3, 38.4, 50.1, 62.1, 81.1, 128.5, 128.8,
128.9, 129.4, 129.5, 129.9, 137.0, 139.3, 156.4, 160.2, 172.9. HRMS
(ESI)m/z calcdforC24H30O3N3 [M+ H]+ 408.2282;found408.2299.
Benzhydrylidene Aza-Phenylalanine-D-Phenylalanine Benzyl
Ester (34b)
This was synthesized from benzophenone aza-Gly-D-Phe-OH 27
(500 mg, 1.29 mmol) according to the procedure described for
dialkylated aza-analog 33 using benzyl bromide. After purification
by flash chromatography using a gradient solvent from 1 : 9 to
1 : 1 ethyl acetate in hexanes, the evaporation of the collected
fractions afforded 34b as a yellow oil in 63% yield (457 mg). Rf 0.62
Benzhydrylidene Aza-Allylglycinyl-Glycine Ethyl Ester (32)
This was synthesized according to the procedure described for
aza-allylglycinyl dipeptide 29b from the semicarbazone aza-Gly-
Gly-OEt (50 mg, 0.15 mmol) using allyl bromide (20 µl, 0.18 mmol)
as alkylating agent. After purification by flash chromatography
on silica gel using EtOAc in hexanes (1 : 2) as solvent system, the
evaporationofthecollectedfractionsgavetheallylsemicarbazone
32 as an oil (35 mg, 60%): Rf 0.45 (hexanes : EtOAc 2 : 1); 1H NMR
(CDCl3, 400 MHz): δ 1.23 (3H, t, J = 7 Hz), 3.86 (2H, dd, J = 1.6 Hz,
3.8 Hz), 4.05 (2H, d, J = 5.7 Hz), 4.16 (2H, q, J = 7 Hz), 4.78 (1H,
dd, J = 1 Hz, 10 Hz), 4.92 (1H, dd, J = 1 Hz, 10 Hz) 5.33–5.43 (1H,
m), 6.89 (1H, t, J = 5.5 Hz), 7.18–7.42 (10H, m); 13C NMR (CDCl3,
100 MHz): δ 13.8, 42.2, 47.9, 60.8, 116.3, 127.8, 128.2, 128.3, 128.7,
129.3, 129.6, 132.4, 135.6, 138.4, 157.7, 158.4, 170.3. HRMS (ESI)
m/z calcd for C21H24N3O3 [M + H]+ 366.1813; found 366.1812.
(hexanes : EtOAc 1 : 1); [α]20 35.2 (c 0.97, CHCl3). 1H NMR (CDCl3,
D
400 MHz): δ 3.20–3.25 (2H, m), 4.55 (1H, d, J = 22 Hz), 4.63 (1H, d,
J = 22 Hz), 4.99 (1H, m), 5.19 (2H, m), 6.79 (2H, m), 7.08–7.46 (25H,
m); 13C NMR (CDCl3, 100 MHz): δ 39.1, 50.0, 55.3, 67.8, 127.5, 127.7,
128.0, 128.8, 129.1, 129.2, 129.3, 129.4, 130.0, 130.2, 130.5, 130.6,
136.3, 136.5, 137.1, 137.5, 139.6, 158.1, 159.2, 172.8. HRMS (ESI)
m/z calcd for C37H35O3N3 [M + H]+ 568.2595; found 568.2603.
Benzhydrylidene Aza-Propargylglycinyl-Prolinyl-
Diphenylmethylamide (35)
This was synthesized according to the procedure described for
29a using propargyl bromide (11.3 µl, 0.127 mmol) as alkylating
c
www.interscience.com/journal/psc Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2010; 16: 284–296

SOLUTION-PHASE SUBMONOMER AZA-PEPTIDE AND AZA-DKP SYNTHESIS
agent. After purification by chromatography on silica gel using
1 : 1 EtOAc : hexanes as solvent system, the evaporation of the
collected fractions gave the propargyl semicarbazone 35 as a pale
yellowfoam(30 mg, 48%):Rf 0.46(hexanes : EtOAc1 : 1);[α]20D 196
(c 0.17, CHCl3). 1H NMR (CDCl3, 400 MHz): δ 1.75–1.88 (2H, m), 2.01
(1H, t, J = 2.4 Hz), 2.10–2.17 (2H, m), 3.53–33.62 (3H, m), 4.33 (1H,
dd, J = 2.3 Hz, 17.6 Hz), 4.78 (1H, dd, J = 6 Hz, 7.75 Hz), 6.21 (1H,
d, J = 8.3 Hz), 7.23–7.38 (13H, m), 7.38–7.40 (5H, m), 7.45–7.47
(3H, m), 7.48 (1H, d, J = 8.3 Hz); 13C NMR (CDCl3, 100 MHz): δ 25.2,
28.3, 37.4, 50.1, 56.7, 61.5, 71.5, 78.9, 127.0, 127.1, 127.2, 128.0,
128.3, 128.4, 128.5, 128.7, 129.5, 130.1, 135.4, 137.8, 141.5, 141.9,
161.0, 161.6, 171.0. HRMS (ESI) m/z calcd for C35H34O4N3 [M + H]+
541.2598; found 541.2603.
HRMS (ESI) m/z calcd for C39H37O2N4 [M + H]+ 593.2911; found
593.2928.
Benzhydrylidene Aza-Alaninyl-Proline Diphenylmethylamide
(35d)
Thiswassynthesizedaccordingtotheproceduredescribedfor35a
from tert-butyl ester 31d (70 mg, 0.17 mmol), which gave 11 mg
(12% yield) of 35d: Rf 0.36 (hexanes : EtOAc 1 : 1); [α]20D 166 (c 0.22,
CHCl3). 1H NMR (CDCl3, 400 MHz): δ 1.78–1.89 (2H, m), 2.11–2.17
(2H, m), 2.74 (3H, s), 3.55–3.69 (2H, m), 4.84 (1H, t, J = 6 Hz), 6.24
(1H, d, J = 8 Hz), 7.22–7.33 (20H, m), 7.38 (1H, d, J = 8 Hz); 13C
NMR (CDCl3, 100 MHz): δ 25.0, 28.4, 36.8, 50.1, 56.1, 61.5, 126.9,
126.9, 127.0, 127.1, 127.2, 127.8, 127.9, 128.2, 128.3, 128.4, 128.5,
128.6, 129.1, 129.5, 135.6, 137.9, 141.4, 141.6, 158.0, 161.8, 171.3.
HRMS (ESI) m/z calcd for C33H33O2N4 [M + H]+ 517.2598; found
517.2609.
Benzhydrilidene Aza-Propargylglycinyl-Proline
Diphenylmethylamide (35a)
Thiswassynthesizedfromtert-butylester31a(88 mg,0.204 mmol)
dissolved in 10 ml of DCM. This solution was treated with
HCl gas bubbles via Teflon canula. After striring for 2 h, the
reaction was evaporated under reduced pressure and placed
under vacuum for 2 h. The crude salt was dissolved in THF, cooled
to −15 ◦C and treated sequentially with isobutyl chloroformate
(27 µl, 0.204 mmol) and N-methyl morpholine (22 µl, 0.204 mmol).
After 5 min, dibenzylmethyl amine (53 µl, 0.306 mmol) was added
to the reaction mixture, which was stirred at −15 ◦C for 2 h. After
the removal of the volatiles by rotary evaporation under reduced
pressure, the crude was amide was purified by chromatography
on silica gel using 50% EtOAc in hexane as eluent. Evaporation of
the collected fractions afforded 16.7 mg (15% yield) of 35a which
exhibited the same spectral and physical properties of material, as
described earlier.
Benzhydrylidene Aza-Propargylglycinyl-Proline (36a)
This was synthesized from benzophenone aza-propargylGly-Pro-
OMe 29a (27 mg, 0.069 mmol) dissolved in 5 ml of a mixture of
3 : 1 MeOH : H2O at 5 ◦C. To this solution was added 2 eq. of LiOH
(3.3 mg, 0.138 mmol) and the reaction was stirred overnight. After
evaporation, the crude material was diluted with 5 ml of 1 N HCl
and extracted with EtOAc (3 × 10 ml). The organic phase was
dried over Na2SO4 and concentrated in vacuo to give the acid
36a as a yellow foam (23 mg, 89%): Rf 0.64 (EtOAc : AcOH 20 : 1);
[α]20D −168 (c 0.1, CHCl3). 1H NMR (CDCl3, 400 MHz): δ 1.81–1.84
(1H, m), 1.89–2.00 (3H, m), 2.10–2.14 (1H, s), 2.14–2.25 (1H, m),
3.64–3.79 (3H, m), 4.26–4.51 (1H, dd, J = 1.6 Hz, 18 Hz), 4.51
(1H, m), 7.34–7.56 (10H, m); 13C NMR (CDCl3, 100 MHz): δ 24.1,
37.7, 49.2, 71.6, 78.4, 127.8, 127.9, 128.3, 128.4, 128.5, 129.4, 129.7,
130.0, 132.1, 135.1, 137.6, 160.2, 161.7. HRMS (ESI) m/z calcd for
C22H22O3N3 [M + H]+ 376.1656; found 376.1652.
Benzhydrilidene Aza-Allylglycinyl-Proline
Diphenylmethylamide (35b)
This was synthesized according to the procedure described above
for the synthesis of amide 35a using tert-butyl ester 31b (80 mg,
0.18 mmol), which afforded 39.7 mg (40% yield) of 35b: Rf 0.38
Benzhydrylidene Aza-Allylglycinyl-Proline (36b)
Thiswassynthesizedaccordingtotheproceduredescribedfor36a.
Evaporationofthecollectedfractionsgavetheallylsemicarbazone
(hexanes : EtOAc 1 : 1); [α]20 228 (c 0.17, CHCl3). 1H NMR (CDCl3,
D
36b as a pale yellow oil (12.4 mg, 44%): Rf 0.58 (EtOAc : AcOH
400 MHz):δ 1.79–1.86(2H,m),2.11–2.16(2H,m),3.39–3.44(1H,dd,
J = 7.6 Hz,16 Hz),3.54–3.62(2H,m),4.18–4.23(1H,dd,J = 4.4 Hz,
20 Hz), 4.80–4.86(2H, m), 4.96–4.98(1H, d,J = 10.4 Hz), 5.51–5.62
(1H, m), 6.25 (1H, d, J = 8.4 Hz), 7.22–7.42 (17H, m), 7.48–7.50 (3H,
m), 7.60 (1H, d, J = 8.4 Hz); 13C NMR (CDCl3, 100 MHz): δ 25.1, 28.3,
50.0, 50.6, 56.2, 61.2, 117.2, 126.9, 126.9, 127.0, 127.1, 127.8, 128.1,
128.2, 128.3, 128.4, 129.3, 129.7, 133.0, 135.7, 137.8, 141.4, 141.5,
159.9, 161.9, 171.0. HRMS (ESI) m/z calcd for C35H35O2N4 [M + H]+
543.2755; found 543.2771.
1
20 : 1); [α]20 −219 (c 0.148, CHCl3). H NMR (CDCl3, 400 MHz): δ
D
1.73–2.00 (3H, m), 2.32–2.35 (1H, m), 3.58–3.73 (3H, m), 4.09 (1H,
dd, J = 5.2 Hz, 15.9 Hz), 4.50 (1H, m), 4.92 (1H, dd, J = 1.2 Hz,
15.6 Hz), 5.08 (1H, dd, J = 1.1 Hz, 10.3 Hz), 5.63–5.68 (1H, m),
7.34–7.37 (4H, m), 7.43–7.53 (6H, m); 13C NMR (CDCl3, 100 MHz):
δ 24.6, 27.8, 29.5, 49.6, 51.6, 61.5, 117.5, 128.1, 128.2, 128.5, 128.6,
128.7, 129.6, 129.9, 130.2, 132.9, 135.7, 137.8, 161.5, 173.6. HRMS
(ESI)m/z calcdforC22H24O3N3 [M+ H]+ 378.1812;found378.1805.
Benzhydrylidene Aza-Phenylalaninyl-Proline (36c)
Benzhydrylidene Aza-Phenylalaninyl-Proline
Diphenylmethylamide (35c)
This was synthesized according to the procedure described for
36a. Evaporation of the collected fractions gave the benzyl
semicarbazone 36c as a pale yellow foam (10.9 mg, 38%): Rf 0.61
(EtOAc : AcOH 20 : 1); [α]20D −154 (c 0.122, CHCl3). 1H NMR (CDCl3,
400 MHz): δ 1.81–2.06 (3H, m), 2.33–2.35 (1H, m), 3.58–3.73 (3H,
m), 4.36 (1H, d, J = 15.7 Hz), 4.51 (1H, m), 4.76 (1H, d, J = 15.7 Hz),
7.00–7.10 (2H, m) 7.12–7.48 (13H, m); 13C NMR (CDCl3, 100 MHz): δ
24.5, 26.4, 27.6, 29.4, 44.9, 49.5, 52.1, 61.5, 126.8, 127.6, 127.7, 127.8,
127.9, 128.4, 128.6, 129.6, 129.7, 130.0, 132.1, 135.2, 136.1, 137.2,
137.6, 161.0, 161.7, 173.6. HRMS (ESI) m/z calcd for C26H26O3N3
[M + H]+ 428.1969; found 428.1963.
Thiswassynthesizedaccordingtotheproceduredescribedfor35a
from tert-butyl ester 31c (50 mg, 0.1 mmol), which afforded 47 mg
(74% yield) of amide 35c: Rf 0.55 (hexanes : EtOAc 1 : 1); [α]20
D
1
240 (c 0.197, CHCl3). H NMR (CDCl3, 400 MHz): δ 1.78–1.90 (2H,
m), 2.14–2.24 (2H, m), 3.57–3.70 (2H, m), 4.06 (1H, d, J = 16 Hz),
4.88–4.92 (1H, m), 4.98 (1H, d, J = 16 Hz), 6.23 (1H, d, J = 8 Hz),
7.02–7.36 (22H, m), 7.38–7.47 (3H, m), 7.57 (1H, d, J = 8 Hz); 13C
NMR (CDCl3, 100 MHz): δ 25.0, 28.3, 50.2, 51.3, 56.7, 61.5, 126.5,
126.8, 126.9, 127.0, 127.6, 127.7, 128.1, 128.2, 128.3, 128.4, 128.5,
129.3, 129.6, 135.6, 136.4, 137.9, 141.3, 141.7, 159.3, 162.1, 171.1.
c
J. Pept. Sci. 2010; 16: 284–296 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

BOURGUET ET AL.
Benzhydrylidene Aza-Alaninyl-Proline (36d)
c-[Aza-Phenylalaninyl-Prolyl] (39c)
Thiswassynthesizedbythetreatmentofbenzophenoneprotected
aza-phenylalaninylproline methyl ester 29c (45 mg, 0.102 mmol)
asdescribedfor39a. Purificationbyflashchromatographyonsilica
gel using 100% EtOAc as solvent provided the aza-DKP 39c as a
pale yellow foam (21.3 mg, 85%): Rf 0.23 (100% EtOAc); [α]20D −38
(c 0.112, CHCl3). 1H NMR (CD3OD, 400 MHz): δ 1.95–2.05 (2H, m),
2.12–2.26 (2H, m), 3.51–3.64 (2H, m), 3.92 (1H, t, J = 7.9 Hz), 4.66
(1H, d, J = 15.05 Hz), 4.78 (1H, d, J = 15 Hz), 7.37 (5H, s); 13C NMR
(CD3OD, 100 MHz) δ 23.8, 27.2, 45.7, 51.1, 58.2, 128.9, 129.0, 129.4,
135.4. HRMS (ESI) m/z calcd for C13H15O2N3 [M + Na]+ 268.1057;
found 268.1069.
This was synthesized according to the procedure described for
36a. Evaporation of the collected fractions gave the methyl
semicarbazone 36d as a light yellow foam (20.2 mg, 57%): Rf
1
0.51 (EtOAc : AcOH 20 : 1); [α]20 −193 (c 0.267, CHCl3). H NMR
D
(CDCl3, 400 MHz): δ 1.81–2.12 (3H, m), 2.18–2.31 (1H, m), 2.81 (3H,
s), 3.68–3.78 (2H, m), 4.58 (1H, m), 7.28–7.53 (10H, m), 7.79–7.82
(1H, m); 13C NMR (CDCl3, 100 MHz): δ 24.6, 28.0, 37.8, 49.9, 61.7,
128.0, 128.1, 128.4, 128.6, 128.7, 129.5, 129.9, 130.1, 132.3, 135.7,
138.0, 159.3, 161.1, 174.2. HRMS (ESI) m/z calcd for C20H22O3N3 [M
+ H]+ 352.1656; found 352.1649.
Aza-Hydantoin 37
Aza-Phenylalaninyl-Proline tert-Butyl Ester (40c)
This was obtained as side product during the synthesis of aza-
propargylglycinyl proline methyl ester 29a, and purified by
chromatography on silica gel using 20% EtOAc in hexanes as
solvent system. Second to be eluted was aza-hydantoin 37, which
gave after evaporation of the collected fractions a white foam
(30.5 mg, 20%): Rf 0.47 (hexanes : EtOAc 1 : 1); [α]20D −166 (c 0.12,
CHCl3). 1H NMR (CDCl3, 400 MHz): δ 1.89–1.94 (2H, m), 2.08–2.19
(1H, m), 3.12–3.23 (1H, m), 3.61 (1H, m), 3.94 (1H, t, J = 8 Hz),
7.33–7.44 (8H, m), 7.71–7.74 (2H, m); 13C NMR (CDCl3, 100 MHz):
δ 27.0, 27.8, 46.5, 62.5, 77.7, 128.4, 128.5, 128.7, 130.2, 130.4,
132.7, 134.9, 136.01, 157.9, 168.4, 180.1. HRMS (ESI) m/z calcd for
C19H18O2N3 [M + H]+ 320.1394; found 320.1398.
Benzhydrylidene aza-phenylalaninyl-glycinyl-proline tert-butyl es-
ter 31c (50 mg, 0.1 mmol) was stirred with hydroxylamine hy-
drochloride (29 mg, 0.42 mmol) in 20 ml of pyridine as solvent
overnight at 60 ◦C. The volatiles were removed by rotary evapora-
tion followed by co-evaporation with DCM and ethyl acetate until
solidification. Purification by flash chromatography on silica gel
using a 1 : 1 mixture of ethyl acetate in hexane afforded amine 40c
(28 mg, 88%) as a pale yellow foam: Rf 0.31 (hexanes : EtOAc 1 : 1);
[α]20D −35 (c 0.1, CHCl3). 1H NMR (CDCl3, 400 MHz): δ 1.44 (9H, s),
1.83–1.95 (3H, m), 2.12–2.19 (1H, m), 3.64–3.67 (2H, m), 4.46 (1H,
m), 4.59 (1H, d, J = 16 Hz), 4.61 (1H, d, J = 16 Hz), 7.28–7.35 (5H,
m); 13C NMR (CDCl3, 100 MHz): δ 23.4, 27.6, 30.1, 49.1, 56.1, 61.7,
80.1, 127.2, 127.3, 128.1, 128.3, 136.3, 172.7. HRMS (ESI) m/z calcd
for C17H26O3N3 [M + H]+ 320.1971; found 320.1969.
c-[Aza-Propargylglycinyl-Prolyl] (39a)
This was synthesized by the treatment of benzophenone pro-
tected aza-propargylglycinyl proline methyl ester 29a (30 mg,
0.077 mmol) with a mixture of 5 ml of HCl 1 N and 5 ml of THF at
40 ◦C overnight. After concentration under reduced pressure, the
aqueous crude mixture was extracted with EtOAc (3 × 10 ml) and
the organic phase was dried over Na2SO4 and concentrated under
vacuum. The crude material was purified by flash chromatography
on silica gel using 100% EtOAc to finally give the aza-DKP 39a as a
pale yellow foam (12.5 mg, 84%): Rf 0.25 (100% EtOAc); [α]20D −18
(c 0.103, CHCl3). 1H NMR (CD3OD, 400 MHz): δ 1.92–2.15 (3H, m),
2.21–2.31 (1H, m), 2.76 (1H, t, J = 2.4 Hz), 3.52 (2H, t, J = 7.2 Hz),
4.11 (1H, dd, J = 7.6 Hz, 9.2 Hz), 4.17 (1H, dd, J = 2.4 Hz, 17.6 Hz),
4.36 (1H, dd, J = 2.4 Hz, 17.6 Hz); 13C NMR (CD3OD, 100 MHz): δ
22.9, 27.4, 37.2, 45.3, 57.7, 73.4, 77.9, 167.3, 175.8. HRMS (ESI) m/z
calcd for C9H12O2N3 [M + H]+ 194.0924; found 194.0931.
(s,s)- and (R, S)-Fmoc-Alaninyl-Aza-Phenylalaninyl-Proline
tert-Butyl Ester (S,S)- and (R,S)-41
L- or D-Fmoc-Alanine (58 mg, 0.188 mmol) was dissolved in THF,
cooledto−15 ◦C, treatedsequentiallywithisobutylchloroformate
(25 µl, 0.188 mmol) and N-methyl morpholine (26 µl, 0.236 mmol),
stirred for 5 min and treated with a solution of aza-phenylalaninyl-
proline tert-butyl ester 40c (50 mg, 0.157 mmol) in a minimum
volume of ethyl acetate (5 ml). After stirring at −15 ◦C for 1 h,
the volatiles were removed under reduced pressure. The crude
material was dissolved in ethyl acetate and washed with brine.
The organic phase was filtered through a pad of silica gel and
evaporated to, respectively, afford crude (S, S)-41 and (R, S)-
41 from which 2 mg of each was used for enantiomeric purity
determination by analytical HPLC. The rest of the crudes was
purified by chromatography on silica gel using a mixture of ethyl
acetate in hexane (1 : 1) to afford 88 mg of (S, S)-41 (92%) and
92 mg of (R, S)-41 (95%) which were analyzed as below.
c-[Aza-Allylglycinyl-Prolyl] (39b)
This was synthesized by the treatment of benzophenone
semicarbazone-protected aza-allylglycinylproline methyl ester
29b (44.2 mg, 0.113 mmol) as described for the synthesis of 39a.
Purification by flash chromatography on silica gel using 100%
EtOAc as solvent provided the aza-DKP 39b as a pale yellow foam
(15.7 mg, 71%): Rf 0.20 (100% EtOAc); [α]20D −54 (c 0.121, CHCl3).
1H NMR (CD3OD, 400 MHz): δ 1.91–1.99 (2H, m), 2.01–2.13 (1H, m),
2.15–2.27 (1H, m), 3.50 (2H, t, J = 6.8 Hz), 3.91 (1H, dd, J = 6 Hz,
16 Hz), 4.05 (1H, dd, J = 7.2 Hz, 8.4 Hz), 4.32 (1H, dd, J = 5.6 Hz,
16 Hz), 5.26 (1H, dd, J = 1.2 Hz, 16.4 Hz), 5.31 (1H, dd, J = 1.2 Hz,
23.6 Hz), 5.86 (1H, m); 13C NMR (CD3OD, 100 MHz): δ 23.1, 27.2,
45.2, 49.1, 57.8, 118.0, 132.4, 154.9, 166.7. HRMS (ESI) m/z calcd for
C9H14O2N3 [M + H]+ 196.1081; found 196.1090.
(S,S)-41
Rf 0.35 (hexane : EtOAc 1 : 1) and 0.32 (hexane : iPrOH 9 : 1); [α]20
D
24 (c 0.133, CHCl3). 1H NMR (CDCl3, 400 MHz): δ 1.26 (3H, d,
J = 7.2 Hz), 1.47 (9H, s), 1.75–1.81 (2H, m), 1.85–1.97 (1H, m),
2.10–2.15 (1H, m), 3.40–3.49 (2H, m), 4.14–4.25 (3H, m), 4.28–4.30
(1H, m), 4.39 (1H, t, J = 7.2 Hz), 4.48 (1H, d, J = 14 Hz), 4.78 (1H, d,
J = 14 Hz), 5.55 (1H, d, J = 7.6 Hz), 7.20–7.35 (7H, m), 7.41 (2H, t,
J = 7.6 Hz), 7.57 (2H, t, J = 6 Hz), 7.77 (2H, d, J = 7.6 Hz), 8.45 (1H,
br); 13C NMR (CDCl3, 100 MHz): δ 18.9, 25.7, 28.4, 29.9, 47.4, 49.3,
53.6, 61.9, 67.6, 77.7, 81.8, 120.4, 125.4, 125.5, 127.5, 128.1, 128.2,
128.8, 129.7, 136.5, 141.5, 144.1, 156.2, 159.7, 171.4, 172.9. HRMS
(ESI)m/z calcdforC35H40O6N4 [M+ H]+ 613.3025;found613.3021.
c
www.interscience.com/journal/psc Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2010; 16: 284–296

SOLUTION-PHASE SUBMONOMER AZA-PEPTIDE AND AZA-DKP SYNTHESIS
(R,S)-41
14 Andre F, Vicherat A, Boussard G, Aubry A, Marraud M. Aza-peptides.
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2004/050689 A2, 2004.
Rf 0.33 (hexane : EtOAc 1 : 1) and 0.26 (hexane : iPrOH 9 : 1); [α]20
D
33(c0.1,CHCl3).1HNMR(CDCl3,400 MHz):δ 1.27(3H,d,J = 7.2 Hz),
1.46(9H,s),1.77–1.82(2H,m),1.89–1.92(1H,m),2.12–2.17(1H,m),
3.48–3.53 (2H, m), 4.13–4.24 (3H, m), 4.28–4.32 (1H, m), 4.39 (1H,
t, J = 6.4 Hz), 4.53 (1H, d, J = 14.4 Hz), 4.72 (1H, d, J = 14.4 Hz),
5.61 (1H, d, J = 7.2 Hz), 7.15–7.24 (3H, m), 7.28–7.33 (5H, m), 7.40
(2H, t, J = 7.6 Hz), 7.56 (2H, d, J = 6.8 Hz), 7.76 (2H, d, J = 7.2 Hz),
8.45 (1H, br); 13C NMR (CDCl3, 100 MHz): δ 18.6, 25.5, 28.4, 29.9,
47.4, 49.3, 53.9, 61.9, 67.6, 77.6, 81.7, 120.4, 125.4, 127.5, 128.1,
128.2, 128.8, 129.6, 136.6, 141.7, 144.1, 156.3, 159.8, 171.4, 172.7.
HRMS (ESI) m/z calcd for C35H40O6N4 [M + H]+ 613.3025; found
613.3021.
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Acknowledgements
The authors thank the Natural Sciences and Engineering Research
Council of Canada (NSERC), the Fonds Que´be´cois de la Recherche
sur la Nature et les Technologies (FQRNT) and the Canadian
Institutes of Health Research (CIP-79848) for the financial support.
We thank Dr Alexandra Fu¨rto¨s of the Universite´ de Montre´al Mass
Spectrometry facility for mass spectral analyses, and Ms Sylvie
Bilodeau and Mr Cedric Malveau of the Regional High-Field NMR
Laboratory for aid with NMR spectroscopy.
23 Gibson C, Goodman SL, Hahn D, Holzemann G, Kessler H. Novel
solid-phase synthesis of azapeptides and azapeptoides via Fmoc-
strategy and its application in the synthesis of RGD-mimetics. J. Org.
Chem. 1999; 64: 7388–7394.
Supporting information
Supporting information may be found in the online version of this
article.
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