Oxyazapeptides: Synthesis, structure determination, and conformational analysis

Article abstract of DOI:10.1021/jo401234g

Herein we report the synthesis, X-ray structure determination, and conformational analysis of a novel class of heteroatom-modified peptidomimetics, which we shall call "oxyazapeptides". Substituting the typical native N-C^α bond with an O-N^α bond creates a completely new, previously unknown family of peptidomimetics, which are hydrolytically stable and display very interesting conformational behavior. Force field calculations revealed that the barrier to rotation around the O-N^α bond in oxyazapeptides is five times lower than that around the N-N^α bond in azapeptides. Also, conformational analysis supported by X-ray suggests that the oxyaza moiety can effectively induce β-turns, which can make the newly discovered oxyazapeptide scaffold a useful tool for drug discovery and for design of biologics.

Full text of DOI:10.1021/jo401234g

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Article
Oxyazapeptides: A New Peptidomimetics Family. Synthesis,
Structure Determination and Conformational Analysis
Suvendu Biswas, Nader E Abo-Dya, Alexander Oliferenko, Amir
Khiabani, Peter James Steel, Khalid A Alamry, and Alan R. Katritzky
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/jo401234g • Publication Date (Web): 24 Jul 2013
Downloaded from http://pubs.acs.org on July 29, 2013
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The Journal of Organic Chemistry
Oxyazapeptides: Synthesis, Structure Determination and Conformational Analysis
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Suvendu Biswas,‡,# Nader E. Abo-Dya,‡,§,# Alexander Oliferenko,‡ Amir Khiabani,‡ Peter
J. Steel,¶ Khalid A. Alamry,$ and Alan R. Katritzky*,‡,$
‡Center for Heterocyclic Compounds, Department of Chemistry, University of Florida, Gainesville, FL
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32611ꢀ7200, USA
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§Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Zagazig University, Zagazigꢀ
44519, Egypt
¶Department of Chemistry, University of Canterbury, Christchurch, New Zealand
$Department of Chemistry, Faculty of Science, King Abdulaziz University, Jeddah, 21589, Saudi
Arabia
katritzky@chem.ufl.edu
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Abstract
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Herein we report synthesis, Xꢀray structure determination and conformational analysis of a novel class
of heteroatomꢀmodified peptidomimetics, which we shall call “oxyazapeptides”. Substituting the typical
native NꢀCα bond with an OꢀNα bond creates a completely new, previously unknown family of
peptidomimetics, which are hydrolytically stable and display very interesting conformational behaviour.
Force field calculations revealed that the barrier to rotation around the OꢀNα bond in oxyazapeptides is
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five times lower than around the NꢀNα bond in azapeptides. Also, conformational analysis supported by
Xꢀray suggests that the oxyaza moiety can effectively induce βꢀturns, which can make the newly
discovered oxyazapeptide scaffold a useful tool for drug discovery and for design of biologics.
Introduction
Peptides and proteins play vital roles in biological and physiological processes. Natural peptides are
widely used as drugs. However, they often need to be modified to circumvent certain problems related
to drug delivery including; (i) affinity for specific receptors, (ii) metabolic stability towards endogenous
proteases, (iii) appropriate bioꢀdistribution and bioꢀavailability and (iv) duration of action.1,2 Such
problems have been addressed by the design of peptidomimetics which may be devoid of many of the
undesirable properties of natural peptides.1 Once the structure of a natural active peptide is known, key
amino acid residues necessary for receptor recognition can be identified by single amino acid
modification of the peptide ligand using novel αꢀsubstituted amino acids and/or amide bond
replacements. The modification of the peptide backbone (Fig. 1A) with a heteroatom leading to
depsipeptides (Fig. 1B)3 and azapeptides (Fig. 1C)4 has proved to be a useful strategy to design
peptidomimetics.
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Fig. 1. General structures of: A Native peptides, B Depsipeptides, C Azapeptides, D Hybrid
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azadepsipeptides, E Oxyazapeptides
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Azapeptides are a peptidomimetics family in which substitution of the easily rotatable CαꢀC(O) bond
in natural peptides by a more rigid urea NαꢀC(O) bond causes significant changes to both the chemical
and the biological properties (Fig. 1C). Azapeptides prefer a limited conformational space with dihedral
angle values close to those of a polyproline II helix and other types of βꢀturns.5ꢀ7 A systematic study
involving sequential replacement of amino acid residues by their azaꢀcounterparts of their effects on
backbone conformation and activity was called ‘‘azaꢀamino acid scanning’’.7ꢀ9
The introduction of an αꢀhydroxy acid into a peptide sequence results in the formation of an ester
bond, also called a depsipeptide bond (Fig. 1B).10 “Amideꢀtoꢀester” substitution is a versatile tool for
investigating the effect of backbone H bonds on the 3D structure formation and stability of proteins.11
Recently, many depsipeptides, such as enniatins12 and cycloocta depsipeptide PF1022A.13 have been
found to be biologically active. Thus, Dyker et al. synthesized a novel class of pseudopeptides named
“azadepsipeptides” (Fig. 1D) and applied the method to the synthesis of a bisꢀaza analogue of the
antiparasitic cyclooctadepsipeptide PF1022A.14
In the present study, we describe de novo design, synthesis, and characterization of oxyazapeptides in
which an amino acid is replaced by an azaꢀhydroxy acid (Fig. 1E). Oxyazapeptides can be considered as
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the depsipeptide analogues of azapeptides where the αꢀamino group of an azaꢀamino acid is replaced by
a hydroxyl group. The effect of “amideꢀtoꢀester” substitution on the wellꢀknown limited conformational
space of azapeptides15,16 was studied computationally. Conformational analysis based on molecular
mechanics calculations revealed that oxyazapeptides should adopt a βꢀturn secondary structure and
enjoy greater conformational freedom which could render them more adaptive to varying steric demand
of biological interactions. Thus insertion of azaꢀhydroxy acid units into biologically active peptides, i.e.
a type of “azaꢀhydroxy acid scan”, would illuminate structure–activity relationships. The newly
developed synthetic protocol was validated by the synthesis of an oxyaza analogue of leuꢀenkephalin, an
endogenous neurotransmitter.
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Results and Discussion
Synthesis of oxyaza di-, tri-, and tetrapeptides
The reaction of hydroxylamine 1a with benzaldehyde afforded the corresponding oxime 2 which was
reduced with sodium cyanoborohydride (NaCNBH3) to give Nꢀbenzylhydroxylamine 3 in 70% overall
yield (Scheme 1).
Scheme 1. Synthesis of Nꢀbenzylhydroxylamine
α−Amino acid ester hydrochloride salts 4a-d were converted into active acyl imidazoles 5a-d by
reaction with carbonyldiimidazole (CDI) in the presence of 2.5 equiv of DIPEA (Hünig’s base) in dry
methylene chloride. Compounds 5a-d were taken to the next step without further purification. Stirring
5a-d with hydroxylamine 1a or Nꢀmethylhydroxylamine 1b or Nꢀbenzylhydroxylamine 3 at 20 oC for 16
h in dry THF containing one equiv of DIPEA afforded free oxyazaꢀdipeptides 6a-f in 85ꢀ92% yields
(Scheme 2, Table 1). No column chromatography was needed to purify the products and a simple
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extractive workꢀup using 2 N HCl gave oxyazaꢀdipeptides displaying satisfactory H and 13C NMR
spectra.
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Scheme 2. Construction of oxyazaꢀdipeptide
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Table 1. Construction of oxyazaꢀdipeptides 6a-f
Entry
R3
H
R1
R2
6,Yielda %
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CH(CH3)2
CH(CH3)2
CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2Ph
C(CH3)3
C(CH3)3
C(CH3)3
CH3
HOꢀAzaꢀGlyꢀValꢀOtBu 6a, 88
HOꢀAzaꢀAlaꢀValꢀOtBu 6b, 90
HOꢀAzaꢀPheꢀValꢀOtBu 6c, 85
HO-AzaAlaꢀLeuꢀOMe 6d, 92
HO-AzaPheꢀLeuꢀOMe 6e, 87
HO-AzaAlaꢀPheꢀOMe 6f, 90
CH3
CH2Ph
CH3
CH2Ph
CH3
CH3
CH3
aIsolated yield
NꢀAcylbenzotriazoles are advantageous reagents to construct peptides, peptidomimetics and peptide
conjugates.17ꢀ20 N−Pg−(αꢀaminoacyl)benzotriazoles 7a-g were prepared following our reported
procedure,17ꢀ19 then coupled with free oxyazaꢀdipeptides 6a-f in dry THF containing one equiv of
DIPEA and catalytic amount of DMAP (Scheme 3, Table 2) to give NꢀPgꢀoxyazaꢀtripeptide esters 8a-j
in 85ꢀ93% yields. In an attempt to show that no racemization occurs during any stage of the reactions,
we also conducted reactions between the CbzꢀAlaꢀBt 7b, 7b + 7b’ (both L and DL forms) and oxyazaꢀ
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dipeptide 6a. The absence of racemization in the oxyazapeptide (8b + 8b’) was deduced from the H
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NMR and the retention of the chirality was further confirmed by chiral HPLC analysis using a (S,S)
WelkꢀO1column (MeCN/H2O 50:50, flow rate 0.15 mL/min, detection at 210 nm). The diastereomer
8b showed a single retentionꢀtime peak at 13.56 min, while its corresponding diastereomeric mixture
(8b + 8b’) showed two well separated peaks at 13.46 and 16.52 min. In our previous studies we also
demonstrated chirality of the reaction is maintained on Nꢀacylation with Nꢀacylbenzotriazoles for
peptides, depsipeptides and azapeptides.17ꢀ19
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Table 2. Preparation of oxyazaꢀtripeptides 8a-j
Entry
RCOBt 7a-g
CbzꢀGlyꢀBt 7a
oxyazaꢀtripeptide 8a-j, Yielda %
CbzꢀGlyꢀOꢀAzaAlaꢀValꢀOtBu 8a, 87
CbzꢀAlaꢀOꢀAzaꢀGlyꢀValꢀOtBu 8b, 87
CbzꢀDLꢀAlaꢀO-AzaGlyꢀValꢀOtBu 8b + 8b’, 90
CbzꢀPheꢀO-AzaAlaꢀLeuꢀOMe 8c, 91
CbzꢀPheꢀO-AzaPheꢀLeuꢀOMe 8d, 90
CbzꢀPheꢀOꢀAzaAlaꢀPheꢀOMe 8e, 95
BocꢀGlyꢀOꢀAzaPheꢀValꢀOtBu 8f, 87
BocꢀβAlaꢀOꢀAzaPheꢀValꢀOtBu 8g, 85
FmocꢀLeuꢀOꢀAzaAlaꢀValꢀOtBu 8h, 89
FmocꢀLeuꢀOꢀAzaPheꢀValꢀOtBu 8i, 93
FmocꢀPheꢀOꢀAzaPheꢀValꢀOtBu 8j, 92
6a-f
6b
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6d
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CbzꢀAlaꢀBt 7b
CbzꢀDLꢀAlaꢀBt 7b + 7b’
CbzꢀPheꢀBt 7c
CbzꢀPheꢀBt 7c
CbzꢀPheꢀBt 7c
6f
BocꢀGlyꢀBt 7d
BocꢀβAlaꢀBt 7e
FmocꢀLeuꢀBt 7f
FmocꢀLeuꢀBt 7f
FmocꢀPheꢀBt 7g
6c
6c
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6c
6c
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Scheme 3. Synthesis of oxyaza triꢀ and tetraꢀpeptides
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Table 3. Preparation of oxyazaꢀtetrapeptides 10a-f
Entry
oxyazaꢀtetrapeptide 10a-f, Yielda %
CbzꢀAlaꢀMetꢀOꢀAzaPheꢀValꢀOtBu 10a, 90
CbzꢀPheꢀMetꢀOꢀAzaAlaꢀValꢀOtBu 10b, 86
CbzꢀAlaꢀMetꢀOꢀAzaAlaꢀLeuꢀOMe 10c, 84
BocꢀGlyꢀGlyꢀOꢀAzaAlaꢀLeuꢀOMe 10d, 87
BocꢀAlaꢀβAlaꢀOꢀAzaPheꢀLeuꢀOMe 10e, 84
BocꢀAlaꢀGabaꢀOꢀAzaAlaꢀValꢀ OtBu 10f, 88
9a-e
6b-e
6c
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CbzꢀAlaꢀMetꢀBt 9a
CbzꢀPheꢀMetꢀBt 9b
CbzꢀAlaꢀMetꢀBt 9a
BocꢀGlyꢀGlyꢀBt 9c
BocꢀAlaꢀβAlaꢀBt 9d
BocꢀAlaꢀGabaꢀBt 9e
6b
6d
6d
6e
6
6b
aIsolated yield
Oxyazapeptide esters 10a-f were prepared in solution by treatment of NꢀPgꢀ(αꢀ
dipeptidoyl)benzotriazoles 9a-e with free oxyazaꢀdipeptides 6b-e in THF containing one equiv of
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DIPEA and a catalytic amount of DMAP for 16 hr at 20 C. All compounds were isolated without
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column chromatography (Scheme 3, Table 3). The target compounds were characterized by H NMR,
13C NMR, and elemental analysis. No detectable racemization of the NꢀPgꢀoxyazaꢀtetrapeptides esters
was observed by chiral HPLC analysis.
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Validation of the synthetic methodology
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We aimed to utilize our methodology and to show general applicability and scope of these “oxyaza”
type novel peptidomimetics by synthesizing a peptidomimetic version of the biologically important
pentapeptide Leuꢀenkephalin.21 Leuꢀenkephalin is an endogenous opioid peptide neurotransmitter found
naturally in the brains of many animals, including humans. Its amino acid sequence is TyrꢀGlyꢀGlyꢀPheꢀ
Leu. Protected tyrosine 11 was first activated and coupled with GlyꢀGly to give tripeptide 12. Then by
using standard benzotriazole methodology the acid group was activated and coupling of compound 13
with free oxyazaꢀdipeptide 6e in dry THF in the presence of 1.0 equiv of DIPEA and 10% catalytic
amount of DMAP gave the target oxyaza analog of Leuꢀenkephalin 14 (Scheme 4).
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Scheme 4. Synthesis of oxyaza analog of Leuꢀenkephalin 14
X-Ray structure determination
It was deemed important to confirm the structure of a representative example of this new
peptidomimetics family. Thus, an Xꢀray crystal structure was determined of compound 8h (Fig. 2),
which crystallizes in the orthorhombic space group P212121. This unambiguously confirmed the
structure and absolute configuration of 8h.
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Fig. 2. Xꢀray crystal structure of 8h
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Conformational analysis
Conformational behaviour of oxyazapeptides would be most interesting to study in comparison with
azapeptides. This can be done by rotations around the common dihedral angles φ and ψ. Here, the angle
φ denotes rotation about the NꢀNα (or OꢀNα) bond, and the angle ψ is rotation about the bond linking the
Nα and the carbonyl carbon (Fig. 3). Rotations around these angles are expected to proceed differently
for azaꢀ and oxyazapeptides, because oxyazapeptides have a set of distinct features such as (i) the
absence of hydrogen at the αꢀatom, (ii) shorter CꢀO and OꢀN bond lengths comparing to CꢀN and NꢀN
bond lengths, and (iii) much less double bond character of the ester CꢀO bond comparing to the amide
CꢀN bond. To make the calculations simpler, model azaꢀ 15 and oxyaza dipeptide 16 were drawn by
cutting the bulky protecting groups and substituting them with methyl groups. These structures were
geometry optimized using the MMX force field (as implemented in the PCModel v. 9.3 software). The
optimized structures are displayed in Fig. 3. One can see that the main difference between 15 and 16 is
the dihedral angle φ, equal to 178° and 103.5° in 15 and 16, respectively. In azapeptide 15, angle φ is
trans due to a certain doubleꢀbonded character of the hydrazine NꢀN bond. By contrast, oxyazapetide 16
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renders a gauche conformation, because rotation around the OꢀN bond is much less hindered.
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Fig. 3. Optimized structures of azapeptide 15 and oxyazapeptide 16
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Energy barriers to rotations can also provide interesting information. To calculate rotational barriers,
the optimized structures 15 and 16 were subjected to the Dihedral Driver procedure, as implemented in
PCModel. The torsional energy plots are shown in Fig. 4. The most striking difference between the azaꢀ
and oxyazapeptide is the barrier to rotation around the N(O)ꢀNα bond. In azapeptide 15, the barrier is as
high as 35 kcal/mol, while in oxyazapeptide 16 it is only 7 kcal/mol. The shape of the potential energy
profile also exhibits a sharp difference: it is a steep double maximum in 15 and a shallow single
maximum in 16. The symmetric maxima on the φ torsional energy plot of 15 occur almost exactly at ꢀ
90° and 90°, while the single maximum of 16 corresponds to a pure cis conformation. In structure 15,
these maxima can be associated with the rehybridization in the hydrazine fragment and a significant
repulsion by the methyl groups, while in 16 the maximum is due to the shorter NꢀO bond. As the
influence of the varying heteroatom drops as the distance increases, rotation around another important
bond described by dihedral angle ψ, N(O)ꢀNαꢀC’ꢀN proceeds in a very similar way in both structures.
As seen in Fig. 4, (right column) the barrier heights (both close to 5 kcal/mol) and the shapes of the
torsional energy profiles are very similar in structures 15 and 16. It was found that inclusion of an
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implicit solvation does not significantly change the conformational behaviour, and the gas phase and
solvation calculations were in a good qualitative agreement. To test this, we capped the free amino
groups in 15 and 16 with acetyls and carried out calculations with the GB/SA model, the results of
which are given in Fig. 4S and 5S of the Supporting Information. Therefore the rest of the
conformational analysis was done with gasꢀphase force field calculations.
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Interestingly, the φ and ψ torsional angles observed in the Xꢀray structure (Fig. 2) of 8h are in
excellent agreement with these gas phase calculations. In the solid state, the φ angle is 91.3(2)o while
,
the ψ angle is 177.9(2)o.
Fig. 4. Torsional energy plots for dihedral angles φ (left) and ψ (right) in azapeptide 15 (upper row) and
oxyazapeptide 16 (lower row)
Computational study of β-turn inducing
It is known6,22 that azaamino acid residues are instrumental in inducing βꢀturns and other helical
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structures in peptides. It is believed that this ability stems from a restricted rotation around the NαꢀC’
bond, which is due to a partial double bonded character of the latter. Conformational analysis of a
simple dipeptide with one azaamino unit (ForꢀAlaꢀazaAlaꢀNH2) revealed that a global minimum
structure was one having a set of dihedral angles consistent with a βꢀturn structural motif.6 As the
oxyaza unit enjoys at least one additional degree of freedom, which is the rotation around the CꢀO ester
bond, one would expect oxyazapeptides to attain a βꢀturn structure with even greater ease. To explore
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the ability of the oxyaza unit to induce a βꢀturn, we ran a full conformational search (MMX force field)
of a model ForꢀAlaꢀAlaꢀNH2 dipeptide in which one of the Cα atoms was replaced by nitrogen and the
adjacent amide N was replaced by oxygen. For consistency, this model oxyazaꢀdipeptide was made
isosteric to ForꢀAlaꢀazaAlaꢀNH2 previously published by Lee et al.6 The conformational search found
that a βꢀturn conformer was the global minimum on the potential energy surface.
Fig. 5. Molecular structures of βꢀturn structures of oxyazapeptide 17 (left) and azapeptide 18 (right); the
C10 hydrogen bonds are indicated by the dotted lines.
This oxyazaꢀdipeptide 17 conformer is displayed in Fig. 5, with the isosteric azadipeptide 18 also
shown for the sake of comparison. The dihedral angles φ1, ψ1 and φ2, ψ2 characterizing rotations in
oxyazaꢀdipeptide 17 are found to be ꢀ67, 109°, 109° and 2°, which is very close to the type II βꢀturn
structure.15,16 As seen in Fig. 5, the isosteric azadipeptide 18 also makes a βꢀturn conformation, but,
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according to the set of the dihedral angles ꢀ77°, 76°, 179°, and ꢀ3.7°, the hydrazine moiety prefers a
trans conformation, which can be explained by the steric preference of the adjacent urea group.
Comparative analysis of hydrogen bond contacts in structures 17 and 18 also adds to an understanding
of the steric consequences of the “amideꢀtoꢀester” replacement. As seen in Fig. 5, an unobstructed C10
(tenꢀmembered intramolecular cycle) hydrogen bond is formed in the oxyazaꢀdipeptide 17, which is
characterized by the O1ꢀH(N4) separation of 2.25 Å and the O1ꢀH(N4)ꢀN4 angle of 167°. In the Xꢀray
structure of oxyaza tripeptide 8h (Fig. 2), the respective contact length is slightly longer (2.66 Å) and
the OꢀHꢀN angle is somewhat smaller (155°), which is most likely due to the steric influence of the
valine’s bulky isopropyl group. In azadipeptide structure 18, the respective separation and angle are
2.61 Å and 154.2°, which render the respective hydrogen bond a weaker one compared to that in 17.
Another structural feature of azapeptides, which oxyazapeptides are free of, is the OꢀH(N2) contact. In
structure 18, this contact (2.10 Å and 135.7°) making a C7 hydrogen bond, makes the structure a γꢀturn
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rather than a βꢀturn. On the other hand, OꢀH(N2) should be a weaker hydrogen bond than O1ꢀH(N4)
(because of the strong angular dependence of hydrogen bond strength) and therefore can be considered
as one exerting an assistance to the primary O1ꢀH(N4) in keeping the βꢀturn conformation. As
distinguished from 18, the βꢀturn conformation of oxyaza structure 17 is supported by a single although
stronger hydrogen bond O1ꢀH(N4). As a result, the increased backbone flexibility and a stronger C10
hydrogen bond in oxyazapeptides can put them on a par with azapeptides or even make better βꢀturn
inducers that can be used in the synthesis of artificial peptidomimetics.
Conclusions
We have designed, synthesized and structurally studied a new family of peptidomimetics termed
“oxyazapeptides” in which the normal NꢀCα bond is replaced by an OꢀNα bond. This class of
compounds are conformationally more labile, due to a lower barrier to rotation around the OꢀN and CꢀO
bonds, compared to those around the NꢀC and CꢀN bonds in native peptides. As a result, oxyazapeptides
enjoy a higher degree of conformational freedom that might make them potentially more adaptable to
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varying steric demand in receptor binding. The conformational analysis suggests that the oxyaza moiety
can effectively induce βꢀturns in peptidomimetics and thus serve a useful synthetic auxiliary in the
design of small peptide based drugs. More importantly, these newly discovered oxyazapeptides can be a
useful tool for drug discovery and for the targeted design of biologics.
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Experimental Section
Melting points were determined on a capillary point apparatus equipped with a digital thermometer and
are uncorrected. NMR spectra were recorded with TMS for H (300 MHz) and 13C (75 MHz) as an
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internal reference. Reaction progress was monitored by thinꢀlayer chromatography (TLC) and visualized
by UV light. DCM was dried and distilled over CaH2, whereas tetrahydrofuran (THF) was used after
distillation over Naꢀbenzophenone. Carbonyldiimidazole (CDI), hydroxylamine 1a, Nꢀ
methylhydroxylamine 1b, sodium cyanoborohydride, benzaldehyde and Lꢀamino methyl/tertꢀbutyl ester
hydrochloride 4a–d were purchased from chemical supply companies and used without further
purification. N−Pg−(αꢀaminoacyl)benzotriazoles 7a–g, NꢀPgꢀ(αꢀdipeptidoyl)benzotriazoles 9a–d and
CbzꢀTyrꢀ(OBn)ꢀGlyꢀGlyꢀBt 13 were prepared according to the literature methods.
Computational details: All calculations were done with the PCModel software ver. 9.3, Serena
Software. The MMX force field was used. The ground state structures were identified through full
conformational searches using the GMMX routine of PCModel. The implicit solvent model used was
GB/SA (Generalized Born/Surface Area) with the analytical method of Still. The solvent dielectric
constant was taken 78.30 (water) and the internal dielectric constant was taken 1, which are default
settings in PCModel. Full conformational search was also done with the GB/SA solvent method. The
torsional potentials were calculated with the Dihedral Driver procedure as implemented in PCModel;
start and final angles were ꢀ180 and 180, respectively, with the step equal to 10°.
General methods for the preparation of oxyaza-dipeptide 6a–f
To a suspension of Lꢀamino methyl/tertꢀbutyl ester hydrochloride 4a–d (1.0 mmol, 1.0 equiv) in DCM
(20 mL) at 20 °C were added 2.5 equiv of DIPEA and CDI (carbonyldiimidazole, 1.1 equiv). The
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reaction mixture was stirred for 3 h at rt, and the organic layer was washed with water (2 × 20 mL),
NaHCO3 (3 × 20 mL) and brine solution (2 × 20 mL). The organic layer was dried over MgSO4 and
evaporated under vacuum to give oily mono substituted imidazole derivative 5a–d. The residue 5a–d
(1.0 equiv) was dissolved in dry THF (20 mL) and reacted with N–alkylhydroxylamine 1a,b or 3 (1.0
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equiv) in the presence of DIPEA (1.0 equiv) at 20 C overnight. The reaction mixture was poured into a
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separatory funnel and washed with water (2 × 20 mL), 2 N HCl (3 × 20 mL), brine solution (2 × 20
mL), dried over MgSO4 and the solvent evaporated under vacuum to give oxyazadipeptide 6a–f.
HO-AzaGly-Val-OtBu (6a). Compound 6a was prepared according to the given general procedure for
6a–f.White microcrystals (0.408 g, 88%); mp 125.0–127.0 C; [α]D20 –10.0 (c 1.0, methanol); H NMR
(300 MHz, CDCl3) δ 8.26 (br s, 1H), 7.30 (s,1H), 6.42 (d, J = 9.0 Hz, 1H), 4.28 (dd, J = 9.0, 4.5 Hz,
1H), 2.24–2.06 (m, 1H), 1.45 (s, 9H), 0.94 (d, J = 6.9 Hz, 3H), 0.90 (d, J = 6.6 Hz, 3H); 13C NMR (75
MHz, CDCl3) δ 171.7, 162.0, 82.3, 58.1, 31.6, 28.2, 19.1, 17.8; HRMS (ESIꢀTOF) m/z: [M + Na]+
Calcd for C10H20N2O4Na 255.1315; Found 255.1326.
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HO-AzaAla-Val-OtBu (6b). Compound 6b was prepared according to the given procedure for 6a.
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White microcrystals (0.443 g, 90%); mp 88.0–91.0 C; [α]D –4.0 (c 1.0, methanol); H NMR (300
MHz, CDCl3) δ 7.89 (s, 1H), 6.37 (d, J = 8.7 Hz, 1H), 4.22 (dd, J = 8.9, 4.7 Hz, 1H), 3.13 (s, 3H), 2.18–
2.11 (m, 1H), 1.46 (s, 9H), 0.95 (d, J = 6.9 Hz, 3H), 0.90 (d, J = 6.9 Hz, 3H); 13C NMR (75 MHz,
CDCl3) δ 172.4, 161.3, 82.2, 58.7, 38.8, 31.4, 28.2, 19.2, 17.9; Anal. Calcd for C11H22N2O4: C, 53.64;
H, 9.00; N, 11.37. Found: C, 53.52; H, 9.27; N, 11.04.
HO-AzaPhe-Val-OtBu (6c). Compound 6c was prepared according to the given procedure for 6a.
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White microcrystals (0.548 g, 85%); mp 90.0–92.0 C; [α]D20 –20.0 (c 1.0, methanol); H NMR (300
MHz, CDCl3) δ 7.30–7.12 (m, 5H), 6.41 (d, J = 9.0 Hz, 1H), 4.64–4.45 (m, 2H), 4.19 (dd, J = 8.7, 4.8
Hz, 1H), 2.16–1.97 (m, 1H), 1.39 (s, 9H), 0.85 (d, J = 6.9 Hz, 3H), 0.81 (d, J = 6.9 Hz, 3H); 13C NMR
(75 MHz, CDCl3) δ 172.0, 160.7, 137.1, 128.9, 128.5, 127.5, 82.1, 77.7, 77.3, 76.9, 58.7, 54.8, 31.6,
28.2, 19.1, 17.9;Anal. Calcd for C17H26N2O4: C, 63.33; H, 8.13; N, 8.69. Found: C, 63.38; H, 8.40; N,
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8.65.
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HO-AzaAla-Leu-OMe (6d). Compound 6d was prepared according to the given procedure for 6a. Low
melting solid (0.402 g, 92%); [α]D20 –26.0 (c 1.0, methanol); 1H NMR (300 MHz, CDCl3) δ 6.23 (d, J =
8.1 Hz, 1H), 4.38ꢀ4.29 (m, 1H), 3.67 (s, 3H), 3.07 (s, 3H), 1.70–1.46 (m, 3H), 0.87 (d, J = 6.3 Hz, 6H);
13C NMR (75 MHz, CDCl3) δ 175.0, 161.0, 52.5, 51.9, 41.3, 38.3, 25.0, 23.1, 21.9; Anal. Calcd for
C9H18N2O4: C, 49.53; H, 8.31; N, 12.84. Found: C, 49.34; H, 8.60; N, 12.46.
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HO-AzaPhe-Leu-OMe (6e). Compound 6e was prepared according to the given procedure for 6a.
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White microcrystals (0.256 g, 87%); mp 114.0–116.0 C; [α]D20 –12.0 (c 1.0, methanol); H NMR (300
MHz, CDCl3) δ 7.38–7.13 (m, 5H), 6.30 (d, J = 8.1 Hz, 1H), 4.60 (d, J = 15.0 Hz, 1H), 4.52 (d, J = 15.0
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Hz, 1H), 4.42–4.34 (m, 1H), 3.65 (s, 3H), 1.70–1.48 (m, 3H), 0.88 (d, J = 6.3 Hz, 6H); C NMR (75
MHz, CDCl3) δ 175.0, 161.0, 52.5, 51.9, 41.3, 38.3, 25.0, 23.1, 21.9; Anal. Calcd for C15H22N2O4: C,
61.21; H, 7.53; N, 9.52. Found: C, 61.30; H, 7.92; N, 9.49.
HO-AzaAla-Phe-OMe (6f). Compound 6f was prepared according to the given procedure for 6a. Low
melting solid (0.454 g, 90%); [α]D20 –20.0 (c 1.0, methanol); 1H NMR (300 MHz, CDCl3) δ 7.74 (s, 1H),
7.38–7.18 (m, 3H), 7.16–7.08 (m, 2H), 6.34 (d, J = 7.8 Hz, 1H), 4.76–4.60 (m, 1H), 3.68 (s, 3H), 3.10–
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3.02 (m, 5H); C NMR (75 MHz, CDCl3) δ 173.3, 160.7, 136.1, 129.3, 128.8, 127.3, 54.4, 52.5, 38.5,
38.4; Anal. Calcd for C12H16N2O4: C, 57.13; H, 6.39; N, 11.10. Found: C, 57.52; H, 6.69; N, 10.73.
General methods for the preparation of oxyaza-tri- and tetra-peptide 8a–j, 10a–f
N−Pg−(αꢀaminoacyl)benzotriazoles 7a–g or NꢀPgꢀ(αꢀdipeptidoyl)benzotriazoles 9a–d (1.0 mmol, 1.0
equiv) was dissolved in dry THF (20 mL) and reacted with oxyazaꢀdipeptides 6a–f (1.0 equiv) in the
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presence of DIPEA (1.0 equiv) and a 10% cat. amount of DMAP at 20 C overnight. The reaction
mixture was poured into a separatory funnel and washed with water (2 × 20 mL), 2 N HCl (3 × 20 mL),
brine solution (2 × 20 mL), dried over MgSO4 and evaporated under vacuum to give oxyazaꢀtripeptide
8a–j or oxyazaꢀtetrapeptide 10a–f which were characterized by 1H, 13C NMR and elemental analysis.
Cbz-Gly-O-AzaAla-Val-OtBu (8a). Compound 8a was prepared according to the given general
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procedure for 8a–j. White microcrystals (0.381 g, 87%); mp 80.0–82.0 C; [α]D –13.0 (c 1.0,
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methanol); H NMR (300 MHz, CDCl3) δ 7.32–7.17 (m, 5H), 6.01 (d, J = 8.4 Hz, 1H), 5.52 (t, J = 5.6
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Hz, 1H), 5.07 (s, 2H), 4.22 (dd, J = 8.4, 4.8 Hz, 1H), 3.99 (d, J = 5.7 Hz, 2H), 3.15 (s, 3H), 2.15ꢀ2.04
(m, 1H), 1.38 (s, 9H), 0.87 (d, J = 6.9 Hz, 3H), 0.84 (d, J = 6.9 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ
171.4, 168.0, 158.8, 156.8, 136.1, 128.7, 128.5, 128.3, 82.1, 67.7, 58.9, 42.2, 38.6, 31.6, 28.2, 19.1,
17.9; Anal. Calcd for C21H31N3O7: C, 57.65; H, 7.14; N, 9.60. Found: C, 57.75; H, 7.52; N, 9.40.
Cbz-Ala-O-AzaGly-Val-OtBu (8b). Compound 8b was prepared according to the given general
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procedure for 8a–j. Low melting solid (0.381 g, 87%); [α]D20 –17.0 (c 1.0, methanol); H NMR (300
MHz, CDCl3) δ 8.69 (s, 1H), 7.30–7.20 (m, 5H), 6.28 (d, J = 8.7 Hz, 1H), 5.69 (d, J = 7.2 Hz, 1H), 5.08
(d, J = 12.0 Hz, 1H), 5.01 (d, J = 12.6 Hz, 1H), 4.45–4.35 (m, 1H), 4.26 (dd, J = 8.7, 4.8 Hz, 1H), 2.25–
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2.00 (m, 1H), 1.48–1.33 (m, 12H), 0.89 (d, J = 6.6 Hz, 3H), 0.85 (d, J = 6.6 Hz, 3H); C NMR (75
MHz, CDCl3) δ 172.1, 171.2, 158.6, 156.2, 136.1, 128.7, 128.4, 128.2, 82.3, 67.4, 58.6, 48.9, 31.6,
28.2, 19.0, 17.9, 15.4; Anal. Calcd for C21H31N3O7: C, 57.65; H, 7.14; N, 9.60. Found: C, 57.50; H,
7.40; N, 9.31.
Cbz-(DL)Ala-O-AzaGly-Val-OtBu (8b + 8b’). Compound 8b + 8b’ was prepared according to the
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given general procedure for 8a–j. Low melting solid (0.394 g, 90%); H NMR (300 MHz, CDCl3) δ
8.64 (s, 1H), 7.35–7.15 (m, 5H), 6.24 (br s, 1H), 5.63–5.52 (m, 1H), 5.16–4.98 (m, 2H), 4.50–4.37 (m,
1H), 4.32–4.20 (m, 1H), 2.20–2.04 (m, 1H), 1.47–1.36 (m, 12H), 0.92–0.82 (m, 6H); 13C NMR (75
MHz, CDCl3) δ 172.0, 171.2, 158.6, 156.2, 136.1, 128.7, 128.4, 128.3, 82.3, 67.6, 67.5, 58.5, 49.1,
48.9, 31.6, 28.2, 19.2, 19.1, 17.8, 15.5.; Anal. Calcd for C21H31N3O7: C, 57.65; H, 7.14; N, 9.60. Found:
C, 57.55; H,7.42; N, 9.22.
Cbz-Phe-O-AzaAla-Leu-OMe (8c). Compound 8c was prepared according to the given general
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procedure for 8a–j. White microcystals (0.455 g, 91%); mp 123.0–125.0 C; [α]D –17.0 (c 1.0,
methanol); 1H NMR (300 MHz, CDCl3) δ 7.40–7.00 (m, 10H), 6.46 (d, J = 8.1 Hz, 1H), 5.31–5.24 (m,
1H), 5.08 (d, J = 12.0 Hz, 1H), 4.99 (d, J = 12.0 Hz, 1H), 4.50–4.32 (m, 2H), 3.68 (s, 3H), 3.10–3.03
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(m, 2H), 2.85 (s, 3H), 1.70–1.55 (m, 3H), 0.87 (d, J = 5.4 Hz, 3H), 0.84 (d, J = 4.8 Hz, 3H); 13C NMR
(75 MHz, CDCl3) δ 173.9, 170.5, 159.0, 156.4, 135.7, 134.6, 129.4, 129.2, 128.8, 128.7, 128.3, 127.9,
67.8, 54.9, 52.4, 52.1, 41.1, 38.0, 37.3, 24.8, 23.2, 21.9; Anal. Calcd for C26H33N3O7: C, 62.51; H, 6.66;
N, 8.41. Found: C, 62.76; H, 6.80; N, 8.56.
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Cbz-Phe-O-AzaPhe-Leu-OMe (8d). Compound 8d was prepared according to the given general
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procedure for 8a–j. Low melting solid (0.518 g, 90%); [α]D20 –41.0 (c 1.0, methanol); H NMR (300
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MHz, CDCl3) δ 7.40–6.95 (m, 15H), 6.67 (d, J = 7.8 Hz, 1H), 5.62 (d, J = 4.5 Hz, 1H), 5.08–4.93 (m,
2H), 4.64 (d, J = 15.0 Hz, 1H), 4.50 (d, J = 15.0 Hz, 1H), 4.29–4.18 (m, 1H), 3.64 (s, 3H), 2.77 (d, J =
7.2 Hz, 2H), 1.70–1.52 (m, 3H), 0.87 (d, J = 5.4 Hz, 3H), 0.86 (d, J = 5.7 Hz, 3H); 13C NMR (75 MHz,
CDCl3) δ 173.7, 170.4, 158.7, 156.7, 135.8, 135.4, 135.1, 129.3, 129.2, 129.2, 128.8, 128.6, 128.4,
128.3, 127.9, 127.8, 67.6, 54.9, 54.8, 52.4, 52.3, 40.9, 36.7, 24.8, 23.2, 21.9; HRMS (ESIꢀTOF) m/z: [M
+ Na]+ Calcd for C32H37N3O7Na 598.2524; Found 598.2550.
Cbz-Phe-O-AzaAla-Phe-OMe (8e). Compound 8e was prepared according to the given general
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procedure for 8a–j. White microcystals (0.507 g, 95%); mp 132.0–134.0 C; [α]D –24.0 (c 1.0,
methanol); 1H NMR (300 MHz, CDCl3) δ 7.33–6.98 (m, 15H), 6.57 (d, J = 7.8 Hz, 1H), 5.57 (d, J = 5.4
Hz, 1H), 5.02 (d, J = 12.3 Hz, 1H), 4.93 (d, J = 12.3 Hz, 1H), 4.63 (q, J = 7.2 Hz, 1H), 4.40 (q, J = 6.8
Hz, 1H), 3.60 (s, 3H), 3.11–2.98 (m, 4H), 2.84 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 172.6, 170.3,
158.6, 156.5, 136.7, 135.9, 134.9, 129.5, 129.4, 129.1, 128.8, 128.6, 128.6, 128.3, 127.8, 127.1, 67.7,
55.0, 54.9, 52.4, 38.1, 38.0, 37.3; Anal. Calcd for C29H31N3O7: C, .65.28; H, 5.86; N, 7.88. Found: C,
65.40; H, 6.11; N, 7.92.
Boc-Gly-O-AzaPhe-Val-OtBu (8f). Compound 8f was prepared according to the given general
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procedure for 8a–j. Low melting solid (0.417 g, 87%); [α]D20 –11.0 (c 1.0, methanol); H NMR (300
MHz, CDCl3) δ 7.35–7.20 (m, 5H), 6.03 (d, J = 8.4 Hz, 1H), 5.24 (t, J = 5.4 Hz, 1H), 4.81 (d, J = 15.3
Hz, 1H), 4.72 (d, J = 15.3 Hz, 1H), 4.27 (dd, J = 8.4, 3.3 Hz, 1H), 3.90–3.66 (m, 2H), 2.20–2.04 (m,
1H), 1.43 (s, 9H), 1.39 (s, 9H), 0.91 (d, J = 6.9 Hz, 3H), 0.87 (d, J = 8.4 Hz, 3H); 13C NMR (75 MHz,
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CDCl3) δ 171.0, 168.4, 158.1, 155.8, 135.2, 128.9, 128.4, 127.8, 81.8, 80.4, 59.0, 54.5, 41.4, 31.2, 28.2,
28.0, 18.8, 17.8; HRMS (ESIꢀTOF) m/z: [M + Na]+ Calcd for C24H37N3O7Na 502.2543; Found
502.2524.
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Boc-βAla-O-AzaPhe-Val-OtBu (8g). Compound 8g was prepared according to the given general
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procedure for 8a–j. Low melting solid (0.420 g, 85%); [α]D20 –20.0 (c 1.0, methanol); H NMR (300
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MHz, CDCl3) δ 7.30–7.20 (m, 5H), 5.76 (d, J = 8.4 Hz, 1H), 5.16 (t, J = 5.7 Hz, 1H), 4.82 (d, J = 15.3
Hz, 1H), 4.69 (d, J = 15.3 Hz, 1H), 4.28 (dd, J = 8.4, 4.5 Hz, 1H), 3.30 (q, J = 6.0 Hz, 2H), 2.50 (dd, J =
6.8, 5.3 Hz, 2H), 2.15ꢀ2.03 (m, 1H), 1.41 (s, 9H), 1.37 (s, 9H), 0.88 (d, J = 6.9 Hz, 3H), 0.82 (d, J = 6.9
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Hz, 3H); C NMR (75 MHz, CDCl3) δ 171.4, 169.9, 158.0, 155.9, 135.3, 128.9, 128.5, 128.0, 82.3,
79.6, 58.5, 54.3, 33.1, 31.6, 31.6, 28.4 28.0, 18.8, 17.7; HRMS (ESIꢀTOF) m/z: [M + Na]+ Calcd for
C25H39N3O7Na 516.2687; Found 516.2680.
Fmoc-Leu-O-AzaAla-Val-OtBu (8h). Compound 8h was prepared according to the given general
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procedure for 8a–j. White microcystals (0.518 g, 89%); mp 113.0–115.0 C; [α]D +22.0 (c 1.0,
methanol); 1H NMR (300 MHz, CDCl3) δ 7.71 (d, J = 7.8 Hz, 2H), 7.52 (d, J = 7.5 Hz, 2H), 7.35 (t, J =
7.4 Hz, 2H), 7.26 (t, J = 7.5 Hz, 2H), 6.29 (d, J = 8.7 Hz, 1H), 5.37 (d, J = 6.6 Hz, 1H), 4.40–4.22 (m,
4H), 4.16 (t, J = 7.2 Hz, 1H), 3.19 (s, 3H), 2.20–2.05 (m, 1H), 1.72–1.55 (m, 3H), 1.41 (s, 9H), 1.00–
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0.80 (m, 12H); C NMR (75 MHz, CDCl3) δ 171.4, 171.4, 159.0, 156.5, 143.8, 141.5, 128.0, 127.3,
125.2, 120.2, 81.9, 67.6, 59.1, 52.0, 47.3, 40.5, 38.4, 31.6, 28.2, 25.1, 22.7, 22.2, 19.1, 18.0; Anal. Calcd
for C32H43N3O7: C, 66.07; H, 7.45; N, 7.22. Found: C, 65.7; H,7.77; N,7.58.
Fmoc-Leu-O-AzaPhe-Val-OtBu (8i). Compound 8i was prepared according to the given general
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procedure for 8a–j. White microcystals (0.612 g, 93%); mp 116.0–119.0 C; [α]D –13.0 (c 1.0,
methanol); 1H NMR (300 MHz, CDCl3) δ 7.76 (d, J = 7.5 Hz, 2H), 7.54 (d, J = 7.2 Hz, 2H), 7.44−7.20
(m, 9H), 6.41 (d, J = 8.4 Hz, 1H), 5.31 (d, J = 6.6 Hz, 1H), 4.95 (d, J = 15.3 Hz, 1H), 4.75 (d, J = 15.3
Hz, 1H), 4.40ꢀ4.30 (m, 3H), 4.22−4.08 (m, 2H), 2.21ꢀ2.10 (m, 1H), 1.47 (s, 9H), 1.42−1.17 (m, 3H),
0.95 (d, J = 6.9 Hz, 3H), 0.88 (d, J = 6.9 Hz, 3H), 0.81 (d, J = 6.3, Hz, 6H); 13C NMR (75 MHz, CDCl3)
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δ 171.3, 171.1, 158.6, 156.3, 143.6, 141.3, 135.3, 128.9, 128.3, 127.8, 127.1, 127.1, 125.0, 120.0, 81.6,
67.4, 59.2, 54.7, 51.7, 47.0, 39.8, 31.3, 28.0, 24.5, 22.4, 21.8, 18.9, 17.9; Anal. Calcd for C38H47N3O7:
C, 69.38; H, 7.20; N, 6.39. Found: C, 69.18; H, 7.40; N, 6.60.
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Fmoc-Phe-O-AzaPhe-Val-OtBu (8j). Compound 8j was prepared according to the given general
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procedure for 8a–j. White microcystals (0.636 g, 92%); mp 107.0–109.0 C; [α]D –21.0 (c 1.0,
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methanol); H NMR (300 MHz, CDCl3) δ 7.76 (d, J = 7.8 Hz, 2H), 7.55–7.44 (m, 2H), 7.40 (t, J = 7.4
Hz, 2H), 7.35–7.22 (m, 10H), 7.11 (d, J = 7.8 Hz, 2H), 6.46 (d, J = 8.7 Hz, 1H), 5.33 (d, J = 5.7 Hz,
1H), 4.70 (s, 2H), 4.40–4.30 (m, 3H), 4.20–4.11 (m, 2H), 2.92–2.75 (m, 2H), 2.20–2.10 (m, 1H), 1.46
(s, 9H), 0.95 (d, J = 6.6 Hz, 3H), 0.88 (d, J = 6.9 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 171.1, 170.2,
158.4, 156.2, 143.6, 141.2, 135.3, 135.0, 129.1, 129.0, 128.3, 127.8, 127.6, 127.1, 127.1, 125.0, 120.0,
81.6, 67.5, 59.2, 54.8, 54.3, 47.0, 36.7, 31.3, 28.0, 19.0, 18.0; HRMS (ESIꢀTOF) m/z: [M + Na]+ Calcd
for C41H45N3O7Na 714.3150; Found 714.3186.
Cbz-Ala-Met-O-AzaPhe-Val-OtBu (10a). Compound 10a was prepared according to the given general
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procedure for 8a–j. White microcystals (0.593 g, 90%); mp 83.0–85.0 C; [α]D –31.0 (c 1.0,
methanol); 1H NMR (300 MHz, CDCl3) δ 7.48–7.14 (m, 11H), 6.46 (d, J = 8.7 Hz, 1H), 5.89 (d, J = 7.8
Hz, 1H), 5.16–4.99 (m, 2H), 4.93 (d, J = 15.3 Hz, 1H), 4.55 (d, J = 14.7 Hz, 1H), 4.40–4.05 (m, 3H),
2.37–2.10 (m, 3H), 2.09–1.71 (m, 5H), 1.43 (s, 9H), 1.28 (d, J = 7.2 Hz, 3H), 0.95 (d, J = 6.9 Hz, 3H),
0.91 (d, J = 6.9 Hz, 3H).; 13C NMR (75 MHz, CDCl3) δ 173.2, 171.7, 169.6, 158.6, 156.5, 136.2, 135.4,
129.0, 128.6, 128.5, 128.3, 128.0, 127.9, 81.8, 67.1, 65.9, 59.3, 54.8, 51.4, 31.4, 29.8, 29.2, 28.1, 19.2,
19.1, 18.1, 15.2; Anal. Calcd for C33H46N4O8S: C, 60.16; H, 7.04; N, 8.50. Found: C, 60.10; H,7.14; N,
8.81.
Cbz-Phe-Met-O-AzaAla-Val-OtBu (10b). Compound 10b was prepared according to the given general
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procedure for 8a–j. White microcystals (0.567 g, 86%); mp 58.0–60.0 C; [α]D –23.0 (c 1.0,
methanol); 1H NMR (300 MHz, CDCl3) δ 7.72 (d, J = 6.3 Hz, 1H), 7.30–6.96 (m, 11H), 6.61–6.52 (m,
1H), 4.98 (d, J = 12.3 Hz, 1H), 4.89 (d, J = 12.3 Hz, 1H), 4.57–4.35 (m, 1H), 4.33–4.14 (m, 2H), 3.11–
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2.85 (m, 5H), 2.41–2.21 (m, 1H), 2.19–1.86 (m, 7H), 1.34 (s, 9H), 0.89 (d, J = 6.9 Hz, 6H); C NMR
(75 MHz, CDCl3) δ 172.1, 171.0, 169.1, 158.5, 156.4, 136.3, 136.1, 129.3, 129.2, 128.3, 127.8, 127.6,
126.7, 81.6, 66.7, 60.2, 58.8, 51.3, 37.8, 37.7, 31.3, 29.7, 29.2, 27.8, 18.8, 17.7, 15.1; Anal. Calcd for
C33H46N4O8S: C, 60.16; H, 7. 40; N, 8.50. Found: C, 59.80; H, 7.14; N, 8.53.
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Cbz-Ala-Met-O-AzaAla-Leu-OMe (10c). Compound 10c was prepared according to the given general
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procedure for 8a–j. Low melting solid (0.466 g, 84%); [α]D20 –36.0 (c 1.0, methanol); H NMR (300
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MHz, CDCl3) δ 7.61 (d, J = 5.4 Hz, 1H), 7.39–7.08 (m, 5H), 6.57 (d, J = 8.1 Hz, 1H), 5.93 (d, J = 7.5
Hz, 1H), 5.01 (s, 2H), 4.48–4.14 (m, 3H), 3.59 (s, 3H), 3.08 (s, 3H), 2.58–2.31 (m, 2H), 2.22–1.87 (m,
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5H), 1.68–1.42 (m, 3H), 1.27 (d, J = 7.2 Hz, 3H), 0.85–0.80 (m, 6H); C NMR (75 MHz, CDCl3) δ
174.3, 173.9, 169.7, 158.9, 156.5, 136.2, 128.7, 128.4, 128.0, 67.2, 52.4, 52.1, 51.7, 50.2, 40.9, 38.1,
30.2, 29.5, 24.8, 23.1, 21.8, 17.9, 15.4; HRMS (ESIꢀTOF) m/z: [M + Na]+ Calcd for C25H38N4O8SNa
577.2303; Found 577.2331.
Boc-Gly-Gly-O-AzaAla-Leu-OMe (10d). Compound 10d was prepared according to the given general
o
procedure for 8a–j. White microcystals (0.376 g, 87%); mp 57.0–59.0 C; [α]D20 –6.0 (c 1.0, methanol);
1H NMR (300 MHz, CDCl3) δ 7.53 (t, J = 4.8 Hz, 1H), 6.58 (d, J = 8.1 Hz, 1H), 5.70 (t, J = 5.7 Hz,
1H), 4.54–4.30 (m, 1H), 4.06 (t, J = 5.3 Hz, 2H), 3.95–3.79 (m, 2H), 3.72 (s, 3H), 3.20 (s, 3H), 1.79–
1.54 (m, 3H), 1.44 (s, 9H), 0.94 (d, J = 6.0 Hz, 3H), 0.93 (d, J = 6.3 Hz, 3H); 13C NMR (75 MHz,
CDCl3) δ 174.4, 171.9, 167.7, 159.0, 156.5, 80.4, 52.5, 52.1, 44.0, 41.2, 40.9, 38.4, 28.5, 24.9, 23.1,
21.7; Anal. Calcd for C18H32N4O8: C, 49.99; H, 7.46; N, 12.95. Found: C, 50.41; H, 7.84; N, 12.58.
Boc-Ala-βAla-O-AzaPhe-Leu-OMe (10e). Compound 10e was prepared according to the given general
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procedure for 8a–j. White microcystals (0.451 g, 84%); mp 61.0–63.0 C; [α]D –23.0 (c 1.0,
methanol); 1H NMR (300 MHz, CDCl3) δ 7.25–7.08 (m, 6H), 6.47 (d, J = 8.4 Hz, 1H), 5.44 (d, J = 7.8
Hz, 1H), 4.80 (d, J = 15.3 Hz, 1H), 4.63–4.42 (m, 2H), 4.11–3.95 (m, 1H), 3.62 (s, 3H), 3.52–3.20 (m,
2H), 2.45–2.28 (m, 2H), 1.60–1.47 (m, 3H), 1.32 (s, 9H), 1.19 (d, J = 7.2 Hz, 3H), 0.84 (d, J = 5.4 Hz,
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3H), 0.81 (d, J = 6.3 Hz, 3H); C NMR (75 MHz, CDCl3) δ 174.4, 173.8, 170.1, 158.4, 155.7, 135.5,
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129.0, 128.6, 128.0, 80.0, 54.3, 52.5, 52.0, 50.1, 41.2, 35.3, 33.0, 28.5, 24.9, 23.1, 21.9, 18.6; Anal.
Calcd for C26H40N4O8: C, 58.19; H, 7.51; N, 10.44. Found: C, 58.18; H, 7.77; N, 10.60.
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Boc-Ala-Gaba-O-AzaAla-Val-OtBu (10f). Compound 10f was prepared according to the given general
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procedure for 8a–j. White microcystals (0.432 g, 86%); mp 124.0–126.0 C; [α]D –6.0 (c 1.0,
methanol); 1H NMR (300 MHz, CDCl3) δ 6.72 (br s, 1H), 6.44 (br s, 1H), 5.64 (br s, 1H), 4.38–4.22 (m,
1H), 4.20–4.06 (m, 1H), 3.54–3.35 (m, 1H), 3.17 (s, 3H), 2.54–2.33 (m, 2H), 2.15 (d, J = 4.2 Hz, 2H),
2.01–1.71 (m, 2H), 1.45 (s, 9H), 1.41 (s, 9H), 1.29 (d, J = 6.9 Hz, 3H), 0.92 (d, J = 7.5 Hz, 3H), 0.88 (d,
J = 6.9 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 173.7, 172.0, 170.7, 159.0, 157.4, 82.1, 80.0, 58.9, 50.4,
38.1, 37.9, 31.8, 28.9, 28.5, 28.2, 24.9, 19.0, 18.5, 18.1; Anal. Calcd for C23H42N4O8: C, 54.96; H, 8.42;
N, 11.15. Found: C, 54.96; H, 8.80; N, 11.08.
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Preparation of oxyaza analog of Leu-enkephalin14. The oxyazaꢀdipeptide 6e (1.0 mmol, 1.0 equiv),
DIPEA (1.0 mmol, 1.0 equiv) and 10% cat. amount of DMAP were dissolved in dry THF. A solution of
CbzꢀTyrꢀ(OBn)ꢀGlyꢀGlyꢀBt 13 (1.0 mmol, 1.0 equiv) in THF (5.0 mL) was added and the mixture
stirred for 16 h at 20 °C. The reaction mixture was monitored by TLC [EtOAc–hexanes (1:2)]. After
completion of the reaction, the solvent was evaporated. The residue was dissolved in EtOAc (30 mL)
and washed with 2 N HCl solution (4 × 10 mL), water (10 mL), and brine (10 mL). The solvent was
dried over MgSO4 and evaporated to give oxyaza analog of Leuꢀenkephalin 14. White microcystals
o
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(0.676 g, 85%); mp 88.0–90.0 C; [α]D20 –7.0 (c 1.0, methanol); H NMR (300 MHz, CDCl3) δ 8.45–
8.32 (m, 2H), 7.56–7.17 (m, 19H), 6.91 (d, J = 8.3 Hz, 2H), 5.05 (s, 2H), 4.95 (s, 2H), 4.65 (d, J = 5.7
Hz, 1H), 4.24 (d, J = 12.3 Hz, 2H), 4.08–3.96 (m, 1H), 3.83–3.75 (m, 3H), 3.62 (s, 3H), 3.37 (s, 1H),
3.00 (dd, J = 15.3, 4.8 Hz, 1H), 2.72 (dd, J = 14.1, 3.3 Hz, 1H), 1.73–1.42 (m, 3H), 0.86 (d, J = 4.8 Hz,
3H), 0.82 (d, J = 5.4 Hz, 3H); 13C NMR (75 MHz, DMSOꢀd6) δ 172.7, 171.6, 169.4, 167.7, 157.4,
156.5, 155.6, 136.8, 136.6, 135.5, 129.9, 128.3, 128.0, 127.9, 127.7, 127.4, 127.3, 127.2, 127.1, 114.0,
68.8, 64.9, 56.1, 53.1, 51.5, 51.2, 41.4, 40.0, 36.2, 33.8, 23.8, 22.5, 20.7; Anal. Calcd for C43H49N5O10:
C, 64.89; H, 6.21; N, 8.80. Found: C, 65.15; H, 6.35; N, 8.75.
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Acknowledgments
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We thank the University of Florida and the Kenan Foundation for financial support. This paper was also
funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under Grant
no. (24ꢀ3ꢀ1432/HiCi). The authors, therefore, acknowledge with thanks DSR technical and financial
support. We thank Dr C. D. Hall and Mr. Wang for helpful discussions.
Supporting Information Available
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1H/13C NMR spectra, HPLC chromatography, crystallographic data, and additional computational data.
This material is available free of charge via the Internet at http://pubs.acs.org.
#S. B and N. E. A. D contributed equally to this work.
Notes
The authors declare no competing financial interest.
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