Synthesis and conformational study of model peptides containing N-substituted 3-aminoazetidine-3-carboxylic acids

Article abstract of DOI:10.1002/ejoc.201301741

Model peptides containing N-substituted 3-aminoazetidine-3-carboxylic acids have been synthesized to investigate the conformational features of these C^α-tetrasubstituted amino acids. The target peptides were designed and prepared by classical synthetic methods in solution, exploiting the convenient N-substituted 3-azidoazetidine-3-carboxylic acids as precursors. The conformational preferences of the newly synthesized peptides were investigated in solution by IR and NMR spectroscopy. It was observed that the 3-aminoazetidine-3-carboxylic acid moiety is likely a β-turn inducer. In addition, an interesting main-chain-to-side-chain hydrogen bond that forms a six-membered pseudo-cycle was detected. It connects the nitrogen (acceptor) of the azetidine ring to the amide NH (donor) of the immediately following residue. This unexpected hydrogen bond increases the number of conformational options offered by N-substituted 3-aminoazetidine-3-carboxylic acids when designing foldamers with new and predictable 3D structures.

Full text of DOI:10.1002/ejoc.201301741

FULL PAPER
DOI: 10.1002/ejoc.201301741
Synthesis and Conformational Study of Model Peptides Containing
N-Substituted 3-Aminoazetidine-3-carboxylic Acids
[a,b]
Alessandro Moretto,[c] Cristina Peggion,[c] Marta De Zotti,[c]
ˇ
Asta Zukauskaite˙,
[b]
[c]
[a]
[a]
ˇ
ˇ
Algirdas Sackus, Fernando Formaggio, Norbert De Kimpe,* and Sven Mangelinckx*
Keywords: Nitrogen heterocycles / Amino acids / Conformation analysis / Peptides
Model peptides containing N-substituted 3-aminoazetidine-
3-carboxylic acids have been synthesized to investigate the
conformational features of these Cα-tetrasubstituted amino
acids. The target peptides were designed and prepared by
classical synthetic methods in solution, exploiting the conve-
nient N-substituted 3-azidoazetidine-3-carboxylic acids as
precursors. The conformational preferences of the newly syn-
thesized peptides were investigated in solution by IR and
ine-3-carboxylic acid moiety is likely a β-turn inducer. In ad-
dition, an interesting main-chain-to-side-chain hydrogen
bond that forms a six-membered pseudo-cycle was detected.
It connects the nitrogen (acceptor) of the azetidine ring to the
amide NH (donor) of the immediately following residue. This
unexpected hydrogen bond increases the number of confor-
mational options offered by N-substituted 3-aminoazetidine-
3-carboxylic acids when designing foldamers with new and
NMR spectroscopy. It was observed that the 3-aminoazetid- predictable 3D structures.
Small-size Cα-tetrasubstituted α-amino acids such as α-
Introduction
aminoisobutyric acid (Aib, 1), 1-aminocyclopropane-1-
carboxylic acid (Ac3c, 2), and 1-aminocyclobutane-1-carb-
oxylic acid (Ac4c, 3; Figure 1) have found a great number
of applications in the field of foldamers and beyond.
The secondary structure of an amino acid sequence is of
essential importance for the biological activity of proteins.[1]
This is one of the factors that nowadays sustains the grow-
ing field of “foldamers”.[2] Short peptides based on pro-
teinogenic amino acids are usually quite flexible. Therefore
it is difficult to investigate their 3D structures and to assess
structure–activity relationships. One way to restrict, induce,
and rigidify the conformation of a peptide, as well as to
reduce its biodegradation, is the use of non-proteinogenic
Cα-tetrasubstituted α-amino acids in which the α-hydrogen
atom of the α-amino acid is replaced by an alkyl (or aryl)
group.[3]
By employing such amino acids, even short peptide se-
quences can be made to adopt stable secondary structures
like β-turns,[4] 2.05-helices,[5] 310-helices, and/or α-helices.[6]
Therefore interest in new Cα-tetrasubstituted α-amino acids
has inrceased in recent years resulting in numerous reports
of such building blocks[7] that may be of great importance
for the design of peptidomimetic drugs.[8]
Figure 1. Chemical structures of Aib (1), Ac3c (2), and Ac4c (3).
Aib (1) and Ac4c (3) are known to form short peptides
with a conformational preference for β-turns and 310-heli-
ces.[3g,9] Aib, although not being a proteinogenic amino
acid, has been found in meteorites[10] and is commonly oc-
curring in peptides produced by microbial sources. Exam-
ples of such peptides are the cyclic peptide chlamydocin,
which contains one Aib residue and displays antibiotic,
neuroleptic, and anticancer properties,[11] and, in particular,
the peptaibiotics,[12] in which Aib can represent up to 50%
of the total number of residues. Owing to the peculiar
stereochemical properties of Aib, the peptaibiotics form
stable helices responsible for membrane disruption by dif-
ferent mechanisms[13] and enzyme recognition.[14] Remarka-
bly, Aib, and likewise all Cα-tetrasubstituted α-amino acids,
imparts an important proteolytic stability on a peptide.[15]
Probably due to their small size, Ac3c and Ac4c are par-
tial GluN1 agonists, capable of binding instead of gly-
cine.[16] The Ac3c residue is present in the antitumor agents
cytotrienins[17] and is incorporated into compounds that are
antagonists or inverse agonists of the bradykinin B1-recep-
[a] Department of Sustainable Organic Chemistry and Technology,
Faculty of Bioscience Engineering, Ghent University,
Coupure Links 653, 9000 Ghent, Belgium
E-mail: norbert.dekimpe@UGent.be
sven.mangelinckx@UGent.be
http://www.ugent.be/bw/doct/en
[b] Institute of Synthetic Chemistry, Kaunas University of
Technology,
Radvilenu˛ pl. 19, 50270 Kaunas, Lithuania
˙
[c] Institute of Biomolecular Chemistry, CNR, Padova Unit,
Department of Chemistry, University of Padova,
Via Marzolo 1, 35131 Padova, Italy
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201301741.
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Synthesis and Conformational Study of Model Peptides
tor,[18] and Ac4c is a structural component in an allosteric
non-nucleoside inhibitor of the hepatitis-C virus (HCV).[19]
On the other hand, numerous applications have been
proposed in the bio-organic and supramolecular fields. Just
to mention a few, all three residues 1–3 have been exploited
for the synthesis of naphthalenediimides, which are used in
self-assembling naphthalenediimide (NDI)-based helical or-
ganic nanotubes,[20] and Aib (1) has been used for the syn-
thesis of homo- and heterometallic clusters[21] and exploited
as an electron-transfer[22] or long-range communication me-
diator.[23]
Thus, driven by the opportunities offered by the afore-
mentioned class of amino acids, we started to explore het-
eroatom-containing constrained analogues, which form an
important area of research in modern synthetic chemistry
at the biological interface.[3h,8c,24] The four-membered-ring
heteroatom-containing Cα-tetrasubstituted α-amino acids
4–6 (Figure 2; Azt, R = H) have already been synthe-
sized,[25] however, the conformational bias induced by these
residues on host peptides has not yet been investigated.
Figure 3. Chemical structures of the tripeptides Z-Azt(R)-Ala-Ala-
OMe 7 and Z-Ala-Azt(R)-Ala-OMe 8 (R = tBu, a; R = tAmyl, b).
cation with aqueous HCl (Scheme 1). As these compounds
are very hydrophilic, acid/base extraction was avoided. In-
stead, lyophilization had to be used and the crude products
10 were used in the next steps without further purification
(purity Ͼ95%).
Figure 2. Chemical structures of known, heteroatom-containing
Ac4c analogues. Peptides containing Azt(R) (6: R = tBu, a; R =
tAmyl, b) are described in this work.
Having independently accomplished the synthesis of N-
substituted 3-aminoazetidine-3-carboxylic acids 6 (R = tBu,
tAmyl),[25b] attempts were made to gain a deeper insight
into their conformational features and applications. In ad-
dition to their potential use as foldamer building blocks,
the 3-aminoazetidine-3-carboxylates 6 may be important
for the synthesis of bioactive compounds, as exemplified for
EGF receptor tyrosine kinase inhibitors,[26] CB1 receptor
antagonists,[27] modulators of the NMDA receptor com-
plex,[25a] and inhibitors of bromodomain-containing pro-
teins.[28] However, to the best of our knowledge, the confor-
mational preferences of the residue 6 have not been studied
up to now.
Scheme 1. Hydrolysis of the ester group of 3-azidoazetidine-3-
carboxylic acid esters 9a–d.
The synthesis of N3-Azt(R)-Ala-Ala-OMe derivatives
12a,b was then investigated. Starting from the known di-
peptide Z-Ala-Ala-OMe,[31] both its free amine and its TFA
salt 11 were prepared by catalytic hydrogenation, the latter
to minimize the undesired cyclization to 3,6-dimethylpiper-
azine-2,5-dione during benzyloxycarbonyl deprotection.
The azido acids 10a,b were coupled with H-Ala-Ala-OMe
(or its TFA salt 11) by the HOAt/EDC activation method
to afford the desired N3-Azt(R)-Ala-Ala-OMe derivatives
12a,b. However, under these conditions, only low yields (23–
25%) of the target products were obtained (Scheme 2).
Results and Discussion
Synthesis
To investigate the conformational features of the azetid-
ine-containing Cα-tetrasubstituted amino acids 6 (R = tBu,
tAmyl), and in particular to verify their ability to promote
β-turns, the tripeptides Z-Azt(R)-Ala-Ala-OMe 7 and Z-
Ala-Azt(R)-Ala-OMe 8 (Figure 3; R = tBu, a; R = tAmyl,
b) were synthesized by classical methods of peptide synthe-
sis in solution, including the very convenient α-azido
carboxylic acid intermediates.[29]
To this end, the ester group of the 3-azidoazetidine-3-
carboxylic acid esters 9a–c[25b,30] and 9d (see the Exp. Sect.)
was hydrolyzed in basic medium to quantitatively afford the
hydrochlorides of the free azido acids (10a,b) upon acidifi- Scheme 2. Synthesis of N3-Azt(R)-Ala-Ala-OMe derivatives 12a,b.
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Scheme 3. Improved synthesis of N3-Azt(R)-Ala-Ala-OMe derivatives 12a,b.
Subsequently, the possibility of using the corresponding
As the coupling method via acid chlorides proved to be
acyl chlorides as more reactive intermediates for the cou- the most efficient, it was also used for the synthesis of tri-
pling reaction was investigated. By treatment with oxalyl peptides 8. In situ prepared acid chlorides 13a,b were cou-
chloride,[32] azetidines 10a,b were converted in situ into the pled with H-Ala-OMe to afford dipeptides 15a,b in good
corresponding acyl chlorides 13a,b, which were immediately yields (79–82%). Then catalytic hydrogenation was used to
coupled with H-Ala-Ala-OMe TFA salt 11 under Schotten– reduce the azido compounds 15a,b to the amines 16a,b,
Baumann-like conditions to obtain peptides 12a,b in good which were acylated with the freshly prepared Z-protected
yields (78–80%; Scheme 3).
Finally, reduction of the azido group of N3-Azt(R)-Ala- Ala-OMe 8a,b in good yields (71–75%; Scheme 5).
Ala-OMe 12a,b by catalytic hydrogenation and subsequent In summary, acyl halides, either chlorides or fluorides,
amino acid fluoride 17[33] to give tripeptides Z-Ala-Azt(R)-
treatment of the free primary amine with benzyloxycarb- appear to be the best choice for incorporating the Azt resi-
onyl chloride afforded the target tripeptides Z-Azt(R)-Ala- dues into peptides. However, despite their effectiveness,
Ala-OMe 7a,b in good yields (85–87%; Scheme 4). The Z these activation procedures failed to give satisfactory yields
group was introduced to offer the tripeptide the possibility in the preparation of the sterically hindered homo-Azt(R)
of forming a β-turn as this event requires a hydrogen-bond sequences. To this end, we are currently exploring alterna-
acceptor (the C=O of Z) positioned before Azt.
tive synthetic strategies.
Solution Conformational Analysis
The conformational preferences of the newly synthesized
peptides were investigated to assess whether Azt(R) 6, a Cα-
tetrasubstituted α-amino acid, has β-turn-inducing propen-
sity as well as its optimal position in the β-turn itself. As,
unfortunately, we were unable to grow single crystals suit-
able for X-ray diffraction analysis, the conformational pref-
erences were investigated in CDCl3 solution by IR and
NMR spectroscopy. Circular dichroism did not provide use-
ful information as the peptides are too short and the in-
duced dichroism absorptions of the aromatic Z group over-
lap with those of the amide.
Scheme 4. Synthesis of Z-Azt(R)-Ala-Ala-OMe 7a,b.
Scheme 5. Synthesis of Z-Ala-Azt(R)-Ala-OMe 8a,b.
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Synthesis and Conformational Study of Model Peptides
Figure 4. IR absorption spectra (top lines) of N3-Azt(tBu)-Ala-OMe (15a; left) and N3-Azt(tBu)-Ala-Ala-OMe (12a; right) in CDCl3
solution at 1 mm peptide concentration. The bottom lines are the inverted second derivatives.
Figure 5. IR absorption spectra (top lines) of Z-Azt(tBu)-Ala-Ala-OMe (7a; left) and Z-Ala-Azt(tBu)-Ala-OMe (8a; right) in CDCl3
solution at 1 mm peptide concentration. The bottom lines are the inverted second derivatives.
The two frequency intervals of the IR absorption spec- an influence from the weak hydrogen bond observed in the
trum richest in conformational information were analyzed, shorter peptides (unable to fold into a β-turn, Figure 4) can-
namely 3550–3200 cm–1 (Amide A band), related to the not be excluded.
stretching vibrations of the N–H bonds belonging to the
To ascertain whether the hydrogen bonds are intermo-
urethane and amide groups, and 1800–1600 cm–1 (Amide I lecular or intramolecular, the effect of dilution was studied
band), related to the stretching vibrations of the C=O for all peptides. As an example, Figure 6 shows the spectra
bonds of the ester, urethane, and amide groups. It is gen- of 8a recorded at two concentrations (the 10-fold dilution
erally assumed that solvated NH groups (the so-called free is counterbalanced by a 10-fold increase in the cuvette
NHs) in CDCl3 resonate at wavenumbers higher than pathlength). Only moderate dilution effects were observed
3400 cm–1, whereas hydrogen-bonded NH groups resonate for all peptides, from which it can safely be concluded that
at lower wavenumbers.
the observed hydrogen bonds are of the intramolecular
From the data obtained, at least two types of hydrogen type.
bonds are deduced: One represented by the absorption at
about 3360 cm–1, typical of helix/β-turns, and a second one,
indicative of a weaker hydrogen bond, at about 3400 cm–1.
Indeed, N3-Azt(tBu)-Ala-OMe (15a) and N3-Azt(tBu)-Ala-
Ala-OMe (12a; Figure 4) display an absorption band at
3406 and 3408 cm–1, respectively, that is, borderline between
free (above 3430 cm–1) and strongly hydrogen-bonded (be-
low 3360 cm–1) NHs. As neither peptide forms a β-turn, we
must assume that a nonclassical, weak hydrogen bond is
present.
The tripeptides Z-Azt(R)-Ala-Ala-OMe 7 and Z-Ala-
Azt(R)-Ala-OMe 8 fold into a β-turn, stabilized by a hydro-
gen bond between the urethane C=O and the N–H of the
C-terminal Ala residue. Indeed, an absorption band well
below 3400 cm–1, compatible with a β-turn, is observed in
the IR spectra of all tripeptides 7 and 8, as shown in Fig-
Figure 6. IR absorption spectra of Z-Ala-Azt(tBu)-Ala-OMe (8a)
at 1 mm (A) and 0.1 mm (B) peptide concentrations in CDCl3 solu-
tion.
To summarize, the IR analysis indicates that the two
ure 5 for 7a and 8a. Interestingly, the hydrogen bond of 8a Azt(R) residues investigated are indeed β-turn inducers.
appears to be stronger than that of 7a. Thus, the position However, a weak hydrogen bond competing with that of the
of Azt(R) in the tripeptide sequence is important, even if β-turn appears to interfere with this secondary structure.
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N. De Kimpe, S. Mangelinckx et al.
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The solution conformational analysis was extended to O,O-isopropylidene-α-(hydroxymethyl)serine; a side-chain
NMR spectroscopy. Complete assignment of the reso- oxygen atom acts as hydrogen-bond acceptor for the amide
nances was achieved by means of standard 2D NMR ex- N–H immediately following in the peptide chain, thus clos-
periments. All peptides were subjected to a hydrogen-bond- ing a six-membered pseudo-cycle.[34] Further support for
revealing experiment that involves adding increasing our hypothesis of a side-chain-to-main-chain hydrogen
amounts of deuteriated dimethyl sulfoxide ([D6]DMSO), an bond comes from a recent report in which a trisubstituted
excellent hydrogen-bond acceptor, to a CDCl3 peptide solu- azetidine ring nitrogen participates in a five-membered hy-
tion. The amide protons exposed to the solvent form hydro- drogen-bonded pseudo-cycle.[35]
gen bonds with DMSO, which usually cause a downfield
Also in the case of tripeptides N3-Azt(tBu)-Ala-Ala-
shift of the amide N–H signals. On the other hand, amide OMe (12a) and N3-Azt(tAmyl)-Ala-Ala-OMe (12b), unable
protons that are already engaged in stable hydrogen bonds to fold into a β-turn, an unexpected hydrogen bond is ob-
are shielded from the solvent and thus little affected by vari- served (Figure 7); the Ala2 NH, that is, the NH of residue
ation of the solvent composition. Unexpectedly, DMSO-in- 2, is unaffected by the addition of DMSO, whereas the
sensitive NHs were also found in peptides unable to form chemical shift of the Ala3 NH is quite sensitive to the per-
β-turns, such as the dipeptides N3-Azt(tBu)-Ala-OMe (15a) turbing agent. Here too, a hydrogen bond between the ni-
and N3-Azt(tAmyl)-Ala-OMe (15b; Figure 7). Besides their trogen atom of the azetidine ring of Azt(R) and the amide
insensitivity to the addition of DMSO, the Ala NHs of di- NH of Ala2 may exist. Moving to the Z-protected tripep-
peptides 15a and 15b resonate at unusually low fields. A tides 7 and 8, in which β-turn formation is possible, data
reasonable explanation for these experimental data could be interpretation becomes complex. Indeed, the addition of
the presence of a six-membered pseudo-cycle stabilized by DMSO causes an unexpected upfield shift for the amide
a hydrogen bond between the amide NH immediately fol- NHs following the Azt(R) residues, that is, Ala2 in 7 and
lowing the Azt(R) residue and the trisubstituted nitrogen Ala3 in 8. As an example, Figure 8 shows the behavior of
atom of the azetidine ring. This hypothesis is supported by tripeptides 7a and 8a, the IR absorption spectra of which
a previous, similar finding observed in peptides containing are reported in Figure 5.
1
Figure 7. Plots of the NH chemical shifts in the H NMR spectra of N3-Azt(tBu)-Ala-OMe (15a), N3-Azt(tAmyl)-Ala-OMe (15b), N3-
Azt(tBu)-Ala-Ala-OMe (12a), and N3-Azt(tAmyl)-Ala-Ala-OMe (12b) as a function of increasing percentage of DMSO (vol.-%) in
CDCl3. Peptide concentration: 5 mm.
1
Figure 8. Plots of the H NMR chemical shifts of NH in Z-Azt(tBu)-Ala-Ala-OMe (7a) and Z-Ala-Azt(tBu)-Ala-OMe (8a) as a function
of increasing percentage of DMSO (vol.-%) in CDCl3. Peptide concentration 5 mm.
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Synthesis and Conformational Study of Model Peptides
Figure 9. Sections of the NOESY spectra of Z-Azt(tAmyl)-Ala-Ala-OMe (7b; left) and Z-Ala-Azt(tAmyl)-Ala-OMe (8b; right) showing
the proximity of the tert-amyl group to the amide proton of Ala2 and Ala3, respectively (400 MHz, 2 mm in CDCl3 solution, 298K).
We ascribe the behavior observed in tripeptides 7 and 8 of β-turn conformers is dominant in the equilibrium mix-
to an equilibrium mixture of two hydrogen-bond-stabilized ture, whereas a higher population of the proposed side-
major conformers: a β-turn involving the Z C=O and Ala3 chain-to-main-chain stabilized structure may be present in
NH and a structure, as described above, characterized by a tripeptides 7.
side-chain (azetidine N as acceptor)-to-main-chain (Ala
NH donor) hydrogen bond. Confirmation of the presence
Conclusions
of this latter structure, in addition to the above-mentioned
low-field resonance of the amide NH following Azt(R), is
offered by 2D NMR experiments: The tert-butyl or tert-
amyl groups on the azetidine nitrogen (hydrogen-bond ac-
ceptor) are always close to the amide NH following the
Azt(R) residue. See, for example, the two sections from the
NOESY spectra of Z-Azt(tAmyl)-Ala-Ala-OMe (7b) and
Z-Ala-Azt(tAmyl)-Ala-OMe (8b) reported in Figure 9.
It is also evident that the position (1 or 2) of Azt(R) in
the tripeptide greatly affects the conformation(s) adopted.
Indeed, the IR absorption spectra (Figure 5) show an im-
portant difference in the strength of the hydrogen bonds in
the two tripeptides. In particular, when Azt(R) is at position
2, the IR pattern is closer to that of a β-turn conformation.
This conclusion is in agreement with the findings of the
DMSO experiments described above: The behavior of tri-
peptides 8 is more similar, compared with 7, to that of a
peptide folded into a β-turn because the NH of the residue
at the third position is the least sensitive to DMSO pertur-
bation (Figure 8). However, we must remember that in
peptides 8 the NH at the third position following the
Azt(R) may be involved in a side-chain-to-main-chain hy-
drogen bond as well.
It can be safely concluded that the Azt(R) amino acids
investigated herein are β-turn inducers, similarly to Ac4c,
the prototype of four-membered-ring α-amino acids. In ad-
dition, the trisubstituted nitrogen of the azetidine ring acts
as a hydrogen-bond acceptor, most probably for the proton
of the Azt(R) carboxyamide function. These two tendencies
of the Azt(R) residues may increase the number of confor-
mational options when designing foldamers with new and
predictable 3D structures. However, it is evident that longer
peptides, and in particular Azt(R) homopeptides, are
needed to clarify the extent of the influence of the two tend-
encies on a 3D peptide structure. To this end, we are cur-
rently exploring improved synthetic strategies to access
longer Azt(R) peptides.
Experimental Section
General Methods: Flame-dried glassware was used for all nonaque-
ous reactions. Commercially available solvents and reagents were
purchased from common chemical suppliers and used without fur-
ther purification, unless stated otherwise. The reaction mixtures
were purified by flash chromatography, which was carried out by
using a glass column filled with silica gel (Acros, particle size
0.035–0.070 mm, pore diameter ca. 6 nm). The reactions were mon-
The conformational differences between peptides 7 and
8 parallel those observed, although in the crystal state, for
Ac4c (3; Figure 1) tripeptides of the same type: Z-Ac4c-Ala- itored by initial TLC analysis on glass-backed silica plates (Merck
Kieselgel, 60 F254, precoated 0.25 mm) or aluminium-foil-backed
silica sheets (Merck, Silica gel 60 F254 or Macherey–Nagel, Alug-
ram® Sil G UV 254), which were developed by using standard visu-
alization techniques or agents: UV fluorescence (254 and 366 nm)
and coloring with permanganate solution and subsequent heating.
The migration rate of the compounds is reported as the retention
factor (Rf). Compounds with boiling points lower than 150 °C were
removed by rotary evaporation (20–30 Torr) whereas a high-
vacuum pump (0.01–2 Torr) was used for removal of high-boiling-
point compounds. N-Ethyl,NЈ-[3-(dimethylamino)propyl]carbodi-
Ala-OMe crystallizes into a type I β-turn whereas Z-Ala-
Ac4c-Ala-OMe prefers the type II β-turn.[9b] An inspection
of molecular models reveals that the side-chain-to-main-
chain hydrogen bond of our Azt(R) peptides is possible
only when Azt(R) and the following residue occupy posi-
tions 1 and 2 of a type II (or IIЈ) β-turn (φ1 = –60°, ψ1 =
+120°; φ2 = +80°, ψ2 = 0°). Such a spatial arrangement may
occur in peptides 7, but is unlikely in peptides 8 in which
Azt(R) occupies position 2 (not position 1) of the β-turn.
Therefore we believe that in tripeptides 8 the contribution imide (EDC) and 7-aza-1-hydroxy-1,2,3-benzotriazole (HOAt)[36]
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N. De Kimpe, S. Mangelinckx et al.
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were from GL Biochem products. The l (= S) configuration of the
amino acid Ala was used (for both Z-Ala-OH and the H-Ala-OMe
HCl salt) and was obtained from Sigma–Aldrich as were all other
chemicals. H-Ala-OMe and Z-Ala-Ala-OMe were synthesized ac-
cording to published procedures.[37] For NMR analysis the com-
pounds were diluted in CDCl3, the chemical shifts quoted in parts
per million (ppm), and referenced to tetramethylsilane (TMS, δ =
the solvent under reduced pressure and subsequent removal of
water by lyophilization afforded crude N3-Azt(tAmyl)-OH (10b),
which was used in the next step without further purification or
characterization.
Pd (21 mg, 10 wt.-% on carbon) and TFA (0.15 mL) were added
to a solution of Z-Ala-Ala-OMe (214 mg, 0.69 mmol) in MeOH
(15 mL). The reaction mixture was stirred under H2 (1 atm) at
room temperature for 30 min and then filtered through Celite®.
The filter cake was washed with small portions of MeOH and the
solvent was evaporated under reduced pressure. Excess TFA was
removed by addition of water (1 mL) and subsequent lyophiliza-
tion. The residual crude H-Ala-Ala-OMe·TFA (11) was charac-
terized by 1H NMR and used immediately in the next step without
further purification.
1
0 ppm) or the residual solvent peak. H (300 MHz) and 13C NMR
(75 MHz) spectra were recorded with a JEOL Eclipse FT 300
NMR spectrometer, 1H (400 MHz) and 13C NMR (101 MHz) spec-
tra were recorded with a Bruker DRX 400 spectrometer, and 1H
(200 MHz) and 13C NMR (50 MHz) spectra were recorded with a
Bruker AC 200 spectrometer at room temperature. Peak assign-
ments were elucidated by using COSY, HSQC, HMQC, TOCSY,
NOESY, DEPT-135, and DEPT-90 techniques when required. IR
spectra of samples in neat form were recorded with a Perkin–Elmer
Spectrum BX spectrometer with an ATR (attenuated total reflec-
The crude N3-Azt(tAmyl)-OH (10b; 48 mg, 0.21 mmol) was dis-
solved in anhydrous CH2Cl2 (2 mL) and cooled to 0 °C. Then
HOAt (29 mg, 0.21 mmol) and EDC⋅HCl (40 mg, 0.21 mmol) were
added and the resulting reaction mixture was stirred for 15 min.
Then H-Ala-Ala-OMe⋅TFA (11; 169 mg, 0.63 mmol) and Et3N
(105 mg, 0.145 mL, 1.04 mmol) were added. The homogeneous
mixture formed was warmed to room temperature and stirred for
12 h. Evaporation of the solvent under reduced pressure and purifi-
cation of the residue by flash chromatography on silica gel (petro-
leum ether/EtOAc, 9:1–7:3) afforded pure compound 12b.
tance) accessory; only selected absorbances (ν
[cm–1]) are re-
˜
max
ported. IR absorption measurements on KBr pellets and in CDCl3
solution were performed with a Perkin–Elmer model 1720 X FT-
IR spectrophotometer, nitrogen flushed, equipped with a sample
shuttle device at a nominal resolution of 2 cm–1, averaging 100
scans. Solvent (baseline) spectra were recorded under the same con-
ditions. Cells with pathlengths of 0.1, 1, and 10 mm (with CaF2
windows) were used. Absorption maxima, shoulders, and partially
overlapping bands were detected with the inverse second derivative
method. LC-MS was performed with Agilent 1100 Series VL (ES,
4000V) or SL (ES, 4000V) equipment by using electron-spray ion-
ization at 4 (positive mode) or 3.5 kV (negative mode) and frag-
mentation at 70 eV with only molecular ions ([MH]+) being re-
ported and intensities quoted as a percentage of the base peak
using either a LC-MS coupling or a direct inlet system. Mass spec-
tra were recorded with an Agilent 1100 series mass spectrometer
using either a direct inlet system (electron spray, 4000 V) or LC-MS
coupling (UV detector) or a Mariner ESI-ToF mass spectrometer
(Perseptive Biosystems). Prior to injection, samples were dissolved
in a 1:1 mixture of water/MeOH containing 0.1% formic acid. Op-
tical rotations were measured with a Perkin–Elmer model 241 pola-
rimeter at the sodium D line wavelength using a cell with an optical
pathlength of 10 cm. Concentrations are expressed in g/100 mL.
[α]2D0 were calculated by using the formula [α]2D0 = α/(cl) in which c
is the concentration (in g/mL) and l is the optical path (in dm).
Spectrophotometric-grade MeOH was used as solvent.
Method B: Crude N3-Azt(tAmyl)-OH (10b), prepared from 9d
(0.21 mmol) as described in Method A, was dissolved in anhydrous
CH2Cl2 (2 mL) and cooled to 0 °C. Subsequently DMF (3 μL) and
oxalyl chloride (66 mg, 0.52 mmol) were added. The reaction mix-
ture was stirred at 0 °C for 1 h and at room temperature for 1 h.
Evaporation of the solvent under reduced pressure on a cold water
bath afforded crude N3-Azt(tAmyl)-Cl (13b), which was immedi-
ately dissolved without further purification in anhydrous CH2Cl2
(2 mL) and cooled to 0 °C. Then H-Ala-Ala-OMe·TFA (11;
347 mg, 1.20 mmol) and Et3N (148 mg, 1.47 mmol) were added.
The homogeneous mixture formed was warmed to room tempera-
ture and stirred for 12 h. Evaporation of the solvent under reduced
pressure and purification of the residue by flash chromatography
on silica gel (petroleum ether/EtOAc, 1:1) afforded pure compound
12b.
Light-yellow oil; yield 23% (method A), 78% (method B). Rf =
0.30 (petroleum ether/EtOAc, 1:1). [α]2D0 = –51.2 (c = 0.5, MeOH).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 0.79 (t, J = 7.4 Hz, 3 H,
CH2CH3), 0.87 [s, 6 H, C(CH3)2], 1.12–1.26 (m, 2 H, CH2CH3),
1.34 (d, J = 7.1 Hz, 3 H, CHCH3), 1.35 (d, J = 6.8 Hz, 3 H,
CHCH3), 3.29 [d, J = 7.0 Hz, 2 H, CH(H)NCH(H)], 3.54 [d, J =
7.1 Hz, 1 H, CH(H)NCH(H)], 3.58 [d, J = 7.1 Hz, 1 H, CH(H)-
NCH(H)], 3.68 (s, 3 H, OCH3), 4.42–4.54 (m, 2 H, 2 CHCH3), 6.80
(br. s, 1 H, NH), 7.91 (br. d, J = 7.6 Hz, 1 H, NH) ppm. 13C NMR
(50 MHz, CDCl3, 25 °C): δ = 8.5 (CH2CH3), 18.2 (CHCH3), 18.5
(CHCH3), 20.3 (CCH3), 20.4 (CCH3), 31.6 (CH2CH3), 48.2
(CHCH3), 49.0 (CHCH3), 52.6 (OCH3), 54.6 [C(CH3)2], 55.6
(CH2NCH2), 59.8 (NCH2C), 170.1 (C=O), 171.5 (C=O), 173.2
Synthetic Procedures
N3-Azt(tAmyl)-OMe (9d): N3-Azt(tAmyl)-OMe (9d) was prepared
from Br-Azt(tAmyl)-OMe[25b,30] (157 mg, 2.42 mmol) in accord-
ance with a literature procedure.[25b] Yellow oil; yield 92%. Rf =
0.41 (petroleum ether/EtOAc, 4:1). 1H NMR (300 MHz, CDCl3,
25 °C): δ = 0.85 (t, J = 7.5 Hz, 3 H, CH2CH3), 0.91 [s, 6 H,
C(CH3)2], 1.27 (q, J = 7.5 Hz, 2 H, CH2CH3), 3.32 [d, J = 8.5 Hz,
2 H, CH(H)NCH(H)], 3.71 [d, J = 8.5 Hz, 2 H, CH(H)NCH(H)],
3.84 (s, 3 H, OCH3) ppm. 13C NMR (75 MHz, CDCl3, 25 °C): δ =
8.6 (CH2CH3), 20.0 [C(CH3)2], 31.5 (CH2CH3), 53.2 (OCH3), 54.4
(CH2NCH2), 54.7 (CCH2CH3), 59.7 (NCH2C), 170.6 (C=O) ppm.
(C=O) ppm. IR (KBr): ν = 3314 (NH), 2116 (N ), 1746 (C=O),
˜
3
1657, 1520 [(C=O)NH] cm–1. MS (ES, pos. mode): m/z (%) =
IR (neat): ν = 2108 (N ), 1737 (C=O) cm–1. MS (ES, pos. mode):
˜
3
369.2092 (100) [M + H]+.
m/z (%) = 227 (100) [M + H]+.
N3-Azt(tBu)-Ala-Ala-OMe (12a): This compound was prepared as
described above for 12b starting from 9c. Light-yellow oil; yield
N3-Azt(tAmyl)-Ala-Ala-OMe (12b)
Method A: LiOH·H2O (26 mg, 0.63 mmol) was added to a solution
25% (method A), 80% (method B). Rf = 0.27 (petroleum ether/
of N3-Azt(tAmyl)-OMe (9d; 50 mg, 0.21 mmol) in MeOH (2 mL) EtOAc, 1:1). [α]2D0 = –41.6 (c = 0.5, MeOH). H NMR (400 MHz,
1
and water (2 mL) at 0 °C and the reaction mixture was stirred at
room temperature for 12 h. Subsequently, the resulting reaction
mixture was neutralized with aq. 2 m HCl solution. Evaporation of
CDCl3, 25 °C): δ = 0.93 [s, 9 H, C(CH3)3], 1.34 (d, J = 7.2 Hz, 3
H, CHCH3), 1.35 (d, J = 6.9 Hz, 3 H, CHCH3), 3.30 [d, J = 7.3 Hz,
2 H, CH(H)NCH(H)], 3.57, 3.61 [each d, each J = 7.3 Hz, each 1
2318
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 2312–2321

Synthesis and Conformational Study of Model Peptides
H, CH(H)NCH(H)], 3.68 (s, 3 H, OCH3), 4.41–4.53 (m, 2 H, 2
CHCH3), 6.73 (d, J = 7.5 Hz, 1 H, NH), 7.77 (d, J = 7.6 Hz, 1 H,
N3-Azt(tBu)-Cl (13a) was dissolved in anhydrous CH2Cl2 (2 mL)
and cooled to 0 °C. Then H-Ala-OMe (108 mg, 1.05 mmol) and
NH) ppm. 13C NMR (101 MHz, CDCl3, 25 °C): δ = 18.2 (CCH3), Et3N (22 mg, 0.21 mmol) were added. The homogeneous mixture
18.5 (CCH3), 24.2 [C(CH3)3], 48.2 (CHCH3), 49.1 (CHCH3), 52.3 formed was warmed to room temperature and stirred for 12 h.
[C(CH3)3], 52.6 (OCH3), 55.6 (CH2NCH2), 59.5 (NCH2C), 170.0
Evaporation of the solvent under reduced pressure and purification
(C=O), 171.6 (C=O), 173.2 (C=O) ppm. IR (KBr): ν = 3309 (NH), of the residue by flash chromatography on silica gel (petroleum
˜
2114 (N3), 1744 (C=O), 1654, 1516 [(C=O)NH] cm–1. MS (ES, pos.
ether/EtOAc, 1:1) afforded pure 15a. Light-yellow oil; yield 79%.
Rf = 0.21 (petroleum ether/EtOAc, 1:1). [α]2D0 = –32.4 (c = 0.5,
MeOH) 1H NMR (400 MHz, CDCl3, 25 °C): δ = 1.01 [s, 9 H,
C(CH3)3], 1.44 (d, J = 7.2 Hz, 3 H, CHCH3), 3.37 [d, J = 7.2 Hz,
2 H, CH(H)NCH(H)], 3.65 [d, J = 7.2 Hz, 1 H, CH(H)NCH(H)],
3.69 [d, J = 7.1 Hz, 1 H, CH(H)NCH(H)], 3.76 (s, 3 H, OCH3),
4.58 (quint., J = 7.2 Hz, 1 H, CHCH3), 7.85 (d, J = 5.1 Hz, 1 H,
mode): m/z (%) = 355.2008 (100) [M + H]+.
Z-Azt(tBu)-Ala-Ala-OMe (7a): Pd (4 mg, 10 wt.-% on carbon) was
added to
a solution N3-Azt(tBu)-Ala-Ala-OMe (12a; 40 mg,
0.11 mmol) in MeOH (10 mL). The reaction mixture was stirred
under H2 (1 atm) at room temperature for 30 min, then filtered
through Celite®, and the filter cake was washed with small portions
of MeOH. The solvent was evaporated under reduced pressure. The
residual crude H-Azt(tBu)-Ala-Ala-OMe (14a; 37 mg, 0.11 mmol)
was dissolved in anhydrous CH2Cl2 (4 mL) and cooled to 0 °C.
Subsequently, benzyl chloroformate (23 mg, 0.14 mmol) and N,N-
diisopropylethylamine (15 mg, 0.11 mmol) were added and the re-
action mixture was stirred at room temperature for 12 h. Evapora-
tion of the solvent under reduced pressure and purification of the
residue by flash chromatography on silica gel (CH2Cl2/MeOH,
20:1) afforded pure compound 7a. Light-yellow oil; yield 85%. Rf
= 0.44 (CH2Cl2/MeOH, 19:1). 1H NMR (400 MHz, CDCl3, 25 °C):
δ = 0.99 [s, 9 H, C(CH3)3], 1.32 (d, J = 7.5 Hz, 3 H, CHCH3), 1.35
(d, J = 6.8 Hz, 3 H, CHCH3), 3.25 [s, 2 H, CH(H)NCH(H)], 3.66
(s, 3 H, OCH3), 4.03 [s, 2 H, CH(H)NCH(H)], 4.38–4.54 (m, 2 H,
2 CHCH3), 5.03 (s, 2 H, OCH2), 6.23 (br. s, 1 H, NH), 6.68 (br. s,
1 H, NH), 7.20–7.32 (m, 5 H, 5 CHarom), 9.13 (s, 1 H, NH) ppm.
13C NMR (101 MHz, CDCl3, 25 °C): δ = 18.0 (CHCH3), 18.4
(CHCH3), 24.9 [C(CH3)3], 48.2 (CHCH3), 49.2 (CHCH3), 52.6
(OCH3), 52.7 [C(CH3)3], 54.7 (CH2NCH2), 54.8 (NCH2C), 66.6
(OCH2), 128.1, 128.2 and 128.6 (5 CHarom), 136.4 (Carom), 155.3
NH) ppm. 13C NMR (101 MHz, CDCl3, 25 °C):
δ = 18.5
(CHCH3), 24.3 [C(CH3)3], 47.8 (CHCH3), 52.3 (OCH3), 52.7
[C(CH3)3], 55.7, 55.8 (CH2NCH2), 59.6 (NCH2C), 169.9 (C=O),
173.3 (C=O) ppm. IR (KBr): ν = 3341 (NH), 2116 (N ), 1745
˜
3
(C=O), 1682, 1520 [(C=O)NH] cm–1. MS (ES, pos. mode): m/z (%)
= 284.1573 (100) [M + H]+.
N3-Azt(tAmyl)-Ala-OMe (15b): Compound 15b was prepared as
described above for 15a starting from 9b. Light-yellow oil; yield
82%. Rf = 0.51 (petroleum ether/EtOAc, 7:3). [α]2D0 = –46.4 (c =
0.5, MeOH).1H NMR (200 MHz, CDCl3, 25 °C): δ = 0.80 (t, J =
7.4 Hz, 3 H, CH2CH3), 0.88 [s, 6 H, C(CH3)2], 1.13–1.27 (m, 2 H,
CH2CH3), 1.37 (d, J = 7.2 Hz, 3 H, CHCH3), 3.29 [d, J = 6.9 Hz,
2 H, CH(H)NCH(H)], 3.53 [d, J = 7.0 Hz, 1 H, CH(H)NCH(H)],
3.59 [d, J = 6.9 Hz, 1 H, CH(H)NCH(H)], 3.69 (s, 3 H, OCH3),
4.52 (quint., J = 7.3 Hz, 1 H, CHCH3), 7.91 (d, J = 7.6 Hz, 1 H,
NH) ppm. 13C NMR (101 MHz, CDCl3, 25 °C): δ = 7.3 (CH2CH3),
17.3 (CHCH3), 19.3 [C(CH3)2], 30.5 (CH2CH3), 47.2 (CHCH3),
51.5 (OCH3), 53.6 (CCH2CH3), 54.6 (CH2NCH2), 58.6 (NCH2C),
168.8 (C=O), 172.1 (C=O) ppm. IR (KBr): ν = 3335 (NH), 2118
˜
(N3), 1746 (C=O), 1684, 1520 [(C=O)NH] cm–1. MS (ES, pos.
(C=O), 171.7 (C=O), 173.2 (C=O), 173.4 (C=O) ppm. IR (KBr): ν
˜
mode): m/z (%) = 298.1860 (100) [M + H+].
= 3314 (NH), 1745 (C=O), 1725, 1655, 1521 [(C=O)NH] cm–1. MS
(ES, pos. mode): m/z (%) = 463.2532 (100) [M + H]+.
Z-Ala-Azt(tBu)-Ala-OMe (8a): Cyanuric fluoride (351 mg,
0.22 mL, 2.6 mmol) and pyridine (126 mg, 1.3 mmol) were added
to a solution of Z-Ala-OH (290 mg, 1.3 mmol) in anhydrous
CH2Cl2 (7 mL) at 0 °C under N2 and stirred at room temperature
for 3 h. The reaction mixture was poured into ice–water (10 mL)
and extracted with CH2Cl2 (3ϫ 10 mL). Drying (MgSO4), fil-
tration, and evaporation of the solvent under reduced pressure af-
forded crude Z-Ala-F (17; 219 mg, 0.97 mmol), which was used in
the next step without further purification or characterization.
Z-Azt(tAmyl)-Ala-Ala-OMe (7b): This compound was prepared as
described above for 7a starting from 12b. Light-yellow oil; yield
87%. Rf = 0.44 (CH2Cl2/MeOH, 19:1). [α]2D0 = –57.0 (c = 0.1,
MeOH). 1H NMR (400 MHz, CDCl3, 25 °C): δ = 0.80 (t, J =
7.4 Hz, 3 H, CH2CH3), 0.94 [s, 6 H, C(CH3)2], 1.18–1.33 (m, 2 H,
CH2CH3), 1.32 (d, J = 7.2 Hz, 3 H, CHCH3), 1.35 (d, J = 7.0 Hz,
3 H, CHCH3), 3.14 [d, J = 14.3 Hz, 2 H, CH(H)NCH(H)], 3.67 (s,
3 H, OCH3), 4.09 [s, 2 H, CH(H)NCH(H)], 4.38–4.54 (m, 2 H, 2
CHCH3), 5.03 (s, 2 H, CH2O), 6.14 (br. s, 1 H, NH), 6.60 (d, J =
6.9 Hz, 1 H, NH), 7.20–7.32 (m, 5 H, 5 CHarom), 9.26 (br. s, 1 H,
NH) ppm. 13C NMR (101 MHz, CDCl3, 25 °C): δ = 8.6 (CH2CH3),
17.9 (CHCH3), 18.5 (CHCH3), 21.1 (CCH3), 21.3 (CCH3), 31.9
(CH2CH3), 48.2 (CHCH3), 49.2 (CHCH3), 52.6 (OCH3), 52.9
(CCH2CH3), 54.5 (CH2NCH2), 54.6 (NCH2C), 66.6 (OCH2),
128.1, 128.2, 128.7 (5 CHarom), 136.4 (Carom), 155.3 (C=O), 171.6
Pd (10 mg, 10 wt.-% on carbon) was added to a solution of N3-
Azt(tBu)-Ala-OMe (15a; 55 mg, 0.19 mmol) in MeOH (10 mL).
The reaction mixture was stirred under H2 (1 bar) at room tempera-
ture for 30 min. Then it was filtered through Celite® and the filter
cake was washed with small portions of MeOH. Evaporation of
the solvent under reduced pressure afforded crude H-Azt(tBu)-Ala-
OMe (16a; 50 mg, 0.19 mmol), which was used immediately in the
next step without further purification or characterization.
(C=O), 173.4 (C=O), 173.5 (C=O) ppm. IR (KBr): ν = 3315 (NH),
˜
1744 (C=O), 1723, 1655, 1520 [(C=O)NH] cm–1. MS (ES, pos.
Z-Ala-F (17; 219 mg, 0.97 mmol) was dissolved in anhydrous
CH2Cl2 (5 mL) and cooled to 0 °C. Then H-Azt(tBu)-Ala-OMe
(16a; 50 mg, 0.19 mmol) and Et3N (118 mg, 1.17 mmol) were
added. The homogeneous mixture was warmed to room tempera-
ture and stirred for 2 h. Then the volatiles were evaporated under
reduced pressure and the residue was purified by flash chromatog-
mode): m/z (%) = 477.2608 (100) [M + H]+.
N3-Azt(tBu)-Ala-OMe (15a): Crude N3-Azt(tBu)-OH (10a), which
was prepared as described above for 10b starting from 9a
(0.21 mmol), was dissolved in anhydrous CH2Cl2 (2 mL) and then
cooled to 0 °C. Subsequently, DMF (3 μL) and oxalyl chloride
(70 mg, 0.56 mmol) were added. The reaction mixture was stirred raphy on silica gel (CH2Cl2/MeOH, 20:1) to afford pure compound
at 0 °C for 1 h and also at room temperature for 1 h. Evaporation
of the solvent under reduced pressure on a cold water bath afforded
crude N3-Azt(tBu)-Cl (13a), which was used immediately without
further purification or characterization.
8a. Light-yellow oil; yield 71%. Rf = 0.30 (CH2Cl2/MeOH, 19:1).
[α]2D0 = –57.2 (c = 0.5, MeOH). 1H NMR (400 MHz, CDCl3, 25 °C):
δ = 1.07 [s, 9 H, C(CH3)3], 1.34 (d, J = 7.3 Hz, 3 H, CHCH3), 1.36
(d, J = 7.3 Hz, 3 H, CHCH3), 3.67 (s, 3 H, OCH3), 4.05 [d, J =
Eur. J. Org. Chem. 2014, 2312–2321
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2319

N. De Kimpe, S. Mangelinckx et al.
FULL PAPER
[4]
M. Crisma, A. Moretto, M. De Zotti, F. Formaggio, B. Kapt-
ein, Q. B. Broxterman, C. Toniolo, Biopolymers 2005, 80, 279–
293.
C. Peggion, A. Moretto, F. Formaggio, M. Crisma, C. Toniolo,
Biopolymers (Pept. Sci.) 2013, 100, 621–636.
7.3 Hz, 2 H, CH(H)NCH(H)], 4.11 [d, J = 7.3 Hz, 2 H, CH(H)-
NCH(H)], 4.46 (quint., J = 7.1 Hz, 1 H, CHCH3), 4.94–5.09 (m, 3
H, OCH2, CHCH3), 5.54 (br. s, 1 H, NH), 7.13–7.32 (m, 5 H, 5
CHarom), 7.81 (br. s, 1 H, NH), 8.62 (s, 1 H, NH) ppm. 13C NMR
(101 MHz, CDCl3, 25 °C): δ = 17.8 (2 CHCH3), 24.3 [C(CH3)3],
48.7 (CHCH3), 51.3 (CH2NCH2), 52.5 (OCH3), 52.9 [C(CH3)3],
54.0 (CH2NCH2), 67.1 (OCH2), 68.8 (CHCH3), 128.1, 128.3, 128.7
(5 CHarom), 136.4 (Carom), 156.3 (C=O), 173.1 (C=O), 173.2 (C=O),
[5]
[6]
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C. H. Martin, G. M. Tsai, H. Ueki, V. A. Soloshonok, J. Org.
Chem. 2003, 68, 6208–6214; d) K. Moriyama, H. Sakai, T. Ka-
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König, Beilstein J. Org. Chem. 2009, 5, No. 5; f) M. Pérez-
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Rodríguez, ARKIVOC 2010, 3, 191–202; g) D. S. Radchenko,
O. O. Grygorenko, I. V. Komarov, Amino Acids 2010, 39, 515–
521; h) V. A. Soloshonok, A. E. Sorochinsky, Synthesis 2010,
2319–2344; i) Y. Ohfune, T. Shinada, Eur. J. Org. Chem. 2005,
5127–5143.
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173.3 (C=O) ppm. IR (KBr): ν = 3317 (NH), 1734 (C=O), 1673,
˜
1528 [(C=O)NH] cm–1. MS (ES, pos. mode): m/z (%) = 463.2501
(100) [M + H]+.
[7]
Z-Ala-Azt(tAmyl)-Ala-OMe (8b): This compound was obtained as
described above for 8a starting from 15b. Light-yellow oil; yield
75%. Rf = 0.31 (CH2Cl2/MeOH, 19:1). [α]2D0 = –42.0 (c = 0.5,
MeOH). 1H NMR (400 MHz, CDCl3, 25 °C): δ = 0.88 (t, J =
7.4 Hz, 3 H, CH2CH3), 1.03 [s, 6 H, C(CH3)2], 1.35 (q, J = 7.3 Hz,
2 H, CH2CH3), 1.39 (d, J = 7.1 Hz, 3 H, CHCH3), 1.45 (d, J =
7.2 Hz, 3 H, CHCH3), 3.16–3.42 [m, 2 H, CH(H)NCH(H)], 3.75
(s, 3 H, OCH3), 4.13–4.29 [m, 3 H, CH(H)NCH(H), CHCH3], 4.57
(quint., J = 7.3 Hz, 1 H, CHCH3), 5.07 [d, J = 12.3 Hz, 1 H,
CH(H) O], 5.13 [d, J = 12.3 Hz, 1 H, CH(H)], 5.54 (br. s, 1 H,
NH), 7.16–7.37 (m, 5 H, 5 CHarom), 7.43 (br. s, 1 H, NH), 9.25 (br.
s, 1 H, NH) ppm. 13C NMR (101 MHz, CDCl3, 25 °C): δ = 8.5
(CH2CH3), 18.2 (CHCH3), 18.8 (CHCH3), 21.0, 21.1 [C(CH3)2],
31.6 (CH2CH3), 48.4 (CHCH3), 51.2 (CHCH3), 52.5 (OCH3), 53.0
(CCH2CH3), 53.9 (CH2NCH2), 55.3 (NCH2C), 67.0 (OCH2),
128.1, 128.2, 128.6 (5 CHarom), 136.4 (Carom), 156.0 (C=O), 172.6
[8]
(C=O), 173.1 (C=O) ppm. IR (KBr): ν = 3299 (NH), 1742 (C=O),
˜
[9]
1717, 1666, 1525 [(C=O)NH] cm–1. MS (ES, pos. mode): m/z (%)
= 477.2608 (100) [M + H]+.
Supporting Information (see footnote on the first page of this arti-
1
cle): Mass, H and 13C NMR spectra.
[10]
[11]
[12]
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Acknowledgments
The authors are greatful to the Research Foundation-Flanders
(FWO-Flanders), Ghent University (BOF), Kaunas University of
Technology, Padova University (PRAT), the Italian Ministry of Re-
search (PRIN 2010-2011) and EU COST Action CM0803 (STSM
to A. Z.) for financial support. Dr. Barbara Biondi and Dr. Renato
Schiesari are thanked for their skillful assistance in recording mass
and IR spectra.
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Published Online: February 6, 2014
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