Cyclization of Linear Tetrapeptides Containing N-Methylated Amino Acids by using 1-Propanephosphonic Acid Anhydride

Article abstract of DOI:10.1002/ejoc.201601016

The cyclization of the linear tetrapeptides sequence H-NMePhe-Xaa¹-NMePhe-Xaa²-OH (for which Xaa¹= Xaa²= Ile, Val, or Leu; Xaa¹= Val; Xaa²= Ile or Leu) by using 1-propanephosphonic acid anh

Full text of DOI:10.1002/ejoc.201601016

A Journal of
Accepted Article
Title: Cyclisation of Linear Tetrapeptides Containing N-Methylated Amino Acids
Using 2-Propanephosphonic Acid Anhydride
Authors: Margaret Anne Brimble; Luis De Leon Rodriguez; Allan Zhang; Ernest La-
cey; Andrew Pigott; Ivanhoe Leung
This manuscript has been accepted after peer review and the authors have elected
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of the final Version of Record (VoR). This work is currently citable by using the Digital
Object Identifier (DOI) given below. The VoR will be published online in Early View as
soon as possible and may be different to this Accepted Article as a result of editing.
Readers should obtain the VoR from the journal website shown below when it is pu-
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201601016
Link to VoR: https://doi.org/10.1002/ejoc.201601016
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European Journal of Organic Chemistry
10.1002/ejoc.201601016
FULL PAPER
Cyclisation of Linear Tetrapeptides Containing N-Methylated
Amino Acids Using 2-Propanephosphonic Acid Anhydride
Shengping Zhang,[a] Luis M. De Leon Rodriguez,[b] Ernest Lacey,[c] Andrew M. Piggott, [d] Ivanhoe K. H.
Leung[a] and Margaret A. Brimble*[a,b]
Abstract: The cyclisation of linear tetrapeptides of sequence H-
NMePhe-Xaa1-NMePhe-Xaa2-OH (where Xaa1 = Xaa2 = Ile, Val or
Leu; Xaa1 = Val and Xaa2 = Ile or Leu) using 2-propanephosphonic
acid anhydride is reported. An unanticipated finding was that
cyclisation gives cyclotetrapeptide conformers (kinetic products),
which are highly prone to hydrolysis and that slowly interconvert into
the more stable trans,cis,trans,cis conformers (thermodynamic
products).
Onychocola sclerotica (hereinafter referred to as onychocins),[6]
and more recently, onychocin A (4) and an inseparable complex
of onychocins B (5) and D (7) (Figure 1) were found as the major
metabolites of the fungus, Aspergillus hancockii sp. nov.a The
onychocins showed important activity as cardiac calcium
channel blockers
Endolides A and B, which contain two units of the unusual N-
Me 3-(3-furyl)alanine amino acid, isolated from Stachylidium
sp.[9] and the pseudoxylallemycins, which contain two tyrosines
modified with an allenic ether C-4 building block, from
Pseudoxylaria sp. X802[10] provide more examples of the role of
rare and non-conventional amino acids in the cyclotetrapeptide
scaffold.
Introduction
Cyclotetrapeptides are an uncommon family of microbial
metabolites, with fewer than thirty analogues reported in the
literature. Despite this chemical “under-representation” in nature,
cyclotetrapeptides display diverse pharmacology as phytotoxic
peptides,[1] an inhibitor of lipopolysaccharide-induced nitric oxide
production,[2] antibacterial peptides,[3] inhibitors of histone
deacetylase,[4] inhibitors of glycine transporter type 1[5] and
cardiac calcium channel blockers.[6]
An important class of cyclotetrapeptides involves peptides
containing unnatural N-methylated amino acids (N-Me-aa) within
their sequence. Some examples of these type of peptides are
briefly described below.
Tentoxin (1) (Figure 1), a phytotoxic cyclic tetrapeptide
extracted from the fungus Alternaria alternate, contains a pair of
alternating N-Me-aa, one of which includes a characteristic α,β-
dehydro Phe amino acid.[7]
Hirsutide (2) (Figure 1), a cyclotetrapeptide containing two N-
Me-Phe residues, was isolated from the entomopathogenic
fungus Hirsutella sp. that infected a spider.[8] Additionally, three
cyclotetrapeptides (3-5), structurally related to hirsutide, were
identified from the crude fermentation extract of the fungus
While our understanding of the function of the
cyclotetrapeptides in nature is still evolving, there is a pressing
need for simple synthetic protocols to produce cyclotetrapeptide
scaffolds.
It is well documented that all L-cyclotetrapeptides lacking turn
promoting residues (e.g. proline, D- or N-Me-aa) are difficult to
synthesize given the geometric constraints needed to be
overcome to form the cyclic structure. This can be exemplified
with the tyrosine inhibitor cyclo(Pro-Val-Pro-Tyr),[11] whose
synthesis has been elusive to date despite containing two Pro
residues within its structure. Remarkably, the advent of
methodologies based on ring contraction strategies or the use of
pseudoproline amino acid precursors has provided important
tools
for
the
synthesis
of
the
highly
strained
cyclotetrapeptides.[12] Moreover, even for linear peptides
containing several turn-promoting amino acids, the success of
cyclisation is still sequence-dependent.[13] For instance, the
cyclo(DPro-DPro-Pro-Pro) peptide could only be obtained when
starting from H-DPro-Pro-Pro-DPro-COOH.[14]
Cyclotetrapeptides containing N-Me-aa are particularly
attractive pharmacological leads given their compliance with
Lipinski’s rules, such as not containing more than 5 hydrogen
bond donors and not more than 10 hydrogen bond acceptors.[15]
However, problems frequently encountered during the synthesis
of peptides containing N-Me-aa are: deletions, given the difficult
coupling to the secondary amino group of the N-Me-aa; peptide
bond cleavage (particularly observed for peptides containing
consecutive N-Me-aa)[16] and diketopiperazine formation.[17] Thus,
cyclotetrapeptides containing one or two non-consecutive N-Me-
aa are expected to be the least problematic from a synthetic
point of view and the most stable. Moreover, reports on the
synthesis of these kinds of systems are scarce. In early reports
the synthesis of cyclotetrapeptides containing N-Me-aa was
accomplished by preparing the linear peptide by solution phase
[a]
[b]
S. Zhang, M.A. Brimble
School of Chemical Sciences, The University of Auckland,
23 Symonds St, Auckland, 1142, New Zealand
E-mail: m.brimble@auckland.ac.nz
LM De Leon Rodriguez, M.A. Brimble
Maurice Wilkins Centre for Molecular Biodiscovery, The University of
Auckland
Auckland, 1142, New Zealand
[c]
[d]
E. Lacey
Microbial Screening Technologies
Building AC, 28-54 Percival Rd.,Smithfield, NSW 2164, Australia
A.M. Piggott
Department of Chemistry and Biomolecular Sciences
Macquarie University, NSW 2109, Australia
Supporting information for this article is given via a link at the end of
the document.
a J. Pitt, personal communication, December 2014
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European Journal of Organic Chemistry
10.1002/ejoc.201601016
FULL PAPER
protocols followed by cyclisation of the corresponding 2,4,5-
trichlorophenyl (Tcp) or pentafluorophenyl (Pfp) peptide ester.
Typically, the cyclisation step was carried out under high dilution
conditions in boiling pyridine/solvent mixtures in the presence of
OH (8) precursor of onychocin 6 (synthesized by conventional
solid phase peptide synthesis protocols on a polystyrene resin
derivatized with a 2-chlorotrityl chloride linker (Scheme 1)), using
5 equiv. of T3P, 6 equiv. of DIPEA in CH2Cl2/DMF (1:1) for 24
hours at room temperature at 1 mmol peptide concentration.
Interestingly, the linear peptide dimer and the corresponding
cyclic dimeric octapeptide were the only products observed by
RP-HPLC-UV-ESIMS.
On the other hand, when starting from H-NMePhe-Leu-
NMePhe-Leu-OH (9) under the same conditions using T3P as
the cyclisation agent, the desired cyclic tetrapeptide 6 was
obtained in low yield (15%) together with a small proportion of
the cyclopeptide containing the epimerized C-terminal amino
acid (6*) (Scheme 2 and Figure 2A) (6* was independently
synthesized to enable its thorough characterization). This finding
seems to conflict with the analogous reported synthesis of
hirsutide (2).
base
(e.g.
N,N-dimethyl-4-aminopyridine
and
N,N-
diisopropylethylamine (DIPEA)). However, the yields of the
cyclisation step were usually poor.[18]
The cyclisation step of the linear peptide precursor of tentoxin
(1) has been reported under a variety of conditions, allowing one
to compare the effectiveness of different cyclisation protocols.[19]
Cyclisation yields varied from poor (18%) using the Tcp peptide
ester[19a] to excellent (73% and 81%) when employing the
conventional coupling agents O-(benzotriazol-1-yl)-N,N,N’,N’-
tetramethyluronium hexafluorophosphate (HBTU) and O-(7-
azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium
hexafluorophosphate (HATU) in the presence of base in DMF at
room temperature.[19e]
The synthesis of hirsutide (2) has been reported.[20] The
cyclisation of the Pfp ester of the linear peptide precursor of 2
(H-Phe-NMePhe-Val-NMePhe-OH) was carried out at low
temperature and high peptide concentration giving 2 in high yield
(81%). These conditions are opposite to what has been reported
for other cyclotetrapeptides which were prepared from their
corresponding Pfp ester at high temperature under high dilution
to minimize dimerization.[18]
As already noted the need for new strategies to prepare
cyclotetrapeptides is of paramount importance. In this work the
onychocins were judiciously selected as an informative template
to provide the natural products (3, 4, 5, 7) and closely related
analogue (6) for biological evaluation (Figure 1).
Scheme 1. Initial approach for the synthesis of 6. Reagents and conditions: (i)
Fmoc-N-Me-Phe-OH, CH2Cl2, DIPEA, rt,
1 h; (ii) Fmoc-SPPS (Fmoc
deprotection: 20% piperidine in DMF, rt, 2 × 5 min; coupling: Fmoc-amino acid,
HATU, DIPEA, DMF, rt, 45 min; iii) HFIP/ CH2Cl2 (1:4), rt, 1 h; iv) T3P, CH2Cl2
/DMF (1:1), DIPEA, rt, 24 h.
The yield of 6 was similar to that reported for analogous
systems.[18] Furthermore, the cyclisation was not complete since
the linear peptide precursor 9 and the corresponding C-terminal
epimerized linear peptide 9* were also detected by RP-HPLC
(Scheme 2 and Figure 2A).
Figure 1. N-methylated cyclotetrapeptides
The cyclotetrapeptide 6 was fully characterized by RP-HPLC,
HRMS, NMR (1H, 13C, COSY, HSQC and NOESY), IR (see
supporting information Figures S16-S24) and optical rotation.
The NOESY spectrum of 6 showed a trans configuration for
the N-Me-Phe-Leu peptide bond (CONH) as evidenced by a
correlation between the CH (N-Me-Phe) and NH (Leu) protons
Results and Discussion
We are interested in exploring new methodologies for the
cyclisation of linear tetrapeptides and considered that 2-
propanephosphonic acid anhydride (T3P) may offer advantages
over existing condensing reagents. It is known that T3P gives
superior results compared to HATU with respect to maintaining
the stereochemistry of the cyclisation site of linear pentapeptides
containing sterically hindered amino acids.[21] Additionally, to the
best of our knowledge T3P has not been used in the cyclisation
of linear tetrapeptides. Thus, considering the successful
reported synthesis of hirsutide (2) from its linear precursor H-
Phe-NMePhe-Val-NMePhe-OH,[20] we first proceeded with the
cyclisation of the linear peptide H-Leu-NMePhe-Leu-NMePhe-
(see supporting information Figures S22-S23). Also,
a
correlation between the CH protons of N-Me-Phe and Leu was
observed, which is indicative of a cis configuration for the Leu-N-
Me-Phe peptide bond (CONCH3) (supporting information Figure
S23) (Note that a correlation between CH3 (N-Me-Phe) and CH
(Leu) will indicate a trans configuration).[22]
Therefore, we concluded that 6 exists in a trans,cis,trans,cis
(tctc) conformation in solution, which corresponds to the most
stable structure reported for analogous systems.[23]
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European Journal of Organic Chemistry
10.1002/ejoc.201601016
FULL PAPER
In order to determine optimal conditions for the cyclisation of 9
with T3P, the effect of the solvent, base strength and peptide
concentration was further studied. The cyclisation of 9 to 6 was
observed when the reaction was carried out for 24 hours in a
mixture of CH2Cl2/DMF (1:1) or in CH2Cl2 at room temperature,
although the reaction was slower and low yielding in the later
stages (see supporting information Figure S25). However, when
the reaction was carried out in DMF or EtOAc under similar
conditions no product formation took place but epimerization of
the starting material was observed (see supporting information
Figure S26). Similarly, neither cyclisation nor starting material C-
terminal epimerization occurred with weaker bases such as
collidine or pyridine and only starting material epimerization was
detected using N,N-dimethyl-4-aminopyridine. An increase in the
number of equiv. of T3P or base had no positive effect on the
yield of the desired product but rather a decrease in yield of 6 or
an increase in 9* were observed, respectively. An increase in
the peptide concentration (> 2mM) was detrimental as expected
since an increased of undesired products was determined (e.g.
linear and cyclic dimmers).
(12%) was observed (relative to the proportions observed when
the reaction was carried out at room temperature, 15% and 1%
for 6 and 6* respectively) but 6a, 6b were not detected (Figure
2C). It is important to note that species 6a, 6b were also
detected as minor components of the reaction mixture when the
cyclisation was carried out at room temperature (Figure 2A).
This indicates that 6a and 6b convert into 6 and 6* respectively.
Therefore, one can conclude that in order to drive the reaction
to formation of 6, this has to be carried out either at room or
higher temperatures. This again conflicts with the reported
conditions for the synthesis of hirsutide (2) but agrees with
previous work where cyclisation of activated esters to linear
tetrapeptides was carried out at high temperature.
We next monitored the cyclisation of 9 with T3P at 45 °C as a
function of time (Figure 3, top). Interestingly, the species 6a, 6b
appeared at early reaction times, but disappeared during the
course of the reaction. Moreover, it is worthy to note that the
reaction was not complete using longer reaction times but an
increase in 6* and 9* together with the appearance of several
unidentified impurities was observed (Figure 3). Further
experiments showed that when the cyclisation is done at 0-5 °C
the species 6a, 6b are already present after 1 hour of reaction
(Figure 3, bottom), albeit the proportions of 9* and 6b are less
than 9 and 6a, relative to when the reaction is carried out for
longer times, thus establishing a relationship between these
pairs of species.
9
9*
6
6*
A
6
9*
9
6a 6b
6*
24 hr
B
7 hr
4 hr
6
6a
6b
2 hr
35
6*
20
25
30
C
TIme, min
9*
6
9
24 hr
(45 C)
6*
20
25
30
35
Time,min
Figure 2. RP-HPLC-UV (210 nm) profiles of T3P-mediated cyclisation of 9 at
A) room temperature, B) 0 °C, C) 45 °C; linear gradient of 5%B-95%B over 45
min (ca. 2 %B•min-1), 1 mL•min-1, using XTerra MS C18 column (4.6 mm × 150
mm, 5 µm). A = 0.1% TFA in H2O and B = 0.1 %TFA ACN. Reaction
conditions: 5 equiv. T3P, 6 equiv. DIPEA, DMF/ CH2Cl2 (1:1), 1 day. The scale
bar shown is equivalent to 300 mUA.
3 hr (5 C)
6a
6b
1 hr (5 C)
20
25
30
35
Time, min
Figure 3. (Top graph) RP-HPLC-UV (210 nm) profiles of T3P-mediated
cyclisation of 9 at 45 °C monitored at different times (shown on the right of the
HPLC traces). (Bottom graph) RP-HPLC-UV (210 nm) profiles of T3P-
mediated cyclisation of 9 at 5 °C for 3 h then 45 °C for 24 h monitored at
different times; linear gradient of 5%B-95%B over 45 min (ca. 2 %B•min-1), 1
mL•min-1, using XTerra MS C18 column (4.6 mm × 150 mm, 5 µm). A = 0.1%
TFA in H2O and B = 0.1 %TFA ACN. Reaction conditions: 5 equiv. T3P, 6
equiv. DIPEA, DMF/ CH2Cl2 (1:1). The scale bar shown is equivalent to 500
mUA.
The effect of temperature on cyclisation of 9 with T3P was
next studied. When the reaction was carried out for 24 hours at
low temperature (0-5 °C), a new set of signals (6a, 6b) were
observed by RP-HPLC (Figure 2B) together with 9, 9* and a
small proportion of 6. Remarkably, these signals (6a, 6b)
presented a mass that corresponded to that of the cyclised
product (6) (when analyzed by RP-HPLC-UV-ESIMS, see
supporting information Figure S26), but exhibited shorter
retention times than 6 or 6* (Figure 2). When the reaction was
carried out at 45 °C for 24 hours an increase in the proportion of
6 (53% determined from the RP-HPLC-UV peak area) and 6*
To determine if T3P offers an advantage when compared to
HBTU or HATU, the synthesis of 6 starting from 9 was carried
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European Journal of Organic Chemistry
10.1002/ejoc.201601016
FULL PAPER
out with the latter reagents under similar conditions to those
using T3P, that is, using 5 equiv. of coupling reagent, 6 equiv. of
DIPEA in CH2Cl2/DMF (1:1) for 24 hours at room temperature
and at 1 mmol peptide concentration (see supporting information
Figure S28). Surprisingly, the reaction with HBTU showed the C-
terminal epimerized product 6* as the main cyclic product of the
reaction (~40%, determined from the RP-HPLC-UV peak area),
together with a small amount of 6 (~1%) and the N-formylated
linear peptide (see supporting information Figure S28B).
Interestingly, in the reaction with HATU, 6 was not detected but
6a and 6b were again observed by RP-HPLC (supporting
information Figure S28C). We then performed the reaction with
HATU under similar conditions as stated earlier but at 45 °C.
This time 6* was the major product (~70%, determined from the
RP-HPLC-UV peak area) and 6 was obtained in lesser amount
(~20%) (supporting information Figure S29). These results
further highlight the advantage of T3P to maintain
stereochemical integrity during the cyclisation step.
We next turned our attention to understand the nature of the
species 6a, 6b and for this purpose a couple of hypotheses were
considered. First, one could argue that 6a, 6b correspond to the
activated ester linear intermediates of 9, 9*, if one assumes that
6a, 6b are thermally cyclised in the mass spectrometer[24] but
hydrolyzed to 9, 9* during chromatography. However, 6a, 6b
present the same retention times and molecular masses, when
using either T3P or HATU as activating agents during the
cyclisation of 9. One would have expected different retention
times for the reactive 7-aza-benzotriazol ester of 9 obtained with
HATU relative to the carboxylic phosphonic mixed anhydride of
9 obtained with T3P,[25] which suggests that the hypothesis
proposed above is unlikely.
containing N-Me-aa, an example is the discovery and isolation of
two different conformers of N-Me-D-Ala1-tentoxin analogue of 1
(Figure 1).[26]
Therefore, based on the evidence presented herein we
propose that cyclisation of 9 and 9* with T3P leads to 6a and 6b,
respectively. These peptides (6a and 6b) are cyclic unstable
kinetic products of the reaction and are conformers of 6 and 6*,
respectively, which are the thermodynamic products.
To prove the latter hypothesis we proceeded to identify 6a and
6b, by collecting the corresponding fractions via preparative RP-
HPLC. However, when re-subjecting the purified fractions of 6a
and 6b to RP-HPLC-UV-ESIMS analysis the retention times and
molecular weight of the purified compounds matched those of
the linear peptides 9 and 9* respectively. This indicated that 6a,
6b, presumed to be cyclic peptides have been hydrolysed. While
the ring opening of cyclotetrapeptides has been reported to
occur under relatively mild hydrolytic conditions (Trifluoroacetic
acid (TFA)/H2O, 8:2, 5 hours at temperature),[14a] there is no
precedent for cyclotetrapeptides to undergo ring opening under
slightly acidic conditions such as the those used in RP-HPLC
(mobile phase consists of 0.1% TFA in H2O/ACN mixtures).
Furthermore, attempts to isolate 6a and 6b either by RP-HPLC
under neutral conditions or silica gel chromatography failed even
when the purification was carried out at low temperatures (e.g.
at 5 °C). In both cases only the structures of the linear
precursors 9 and 9* were confirmed after analysis of the attempt
to collect fractions corresponding to 6a and 6b.
We next proceeded to study the cyclisation reaction by NMR.
Therefore, a solution containing 9 at a 2 mM concentration, 1
equiv., of T3P and 2 equiv., of DIPEA in CD2Cl2 was prepared.
Lowering the amounts of DIPEA and T3P was required to
minimize signal overlapping and had no negative effect on the
cyclisation reaction. The reaction mixture was kept at 5 °C for 30
min and an aliquot was taken and analyzed by RP-HPLC and by
HRMS/MS. The reaction mixture was then inserted into the NMR
spectrometer and NMR spectra (1H and 1D NOE) were acquired
at fixed intervals of time while maintaining the reaction
temperature at 5 °C for ~30 min, 27 °C for ~1 hr and 45 °C for
~13.3 hr consecutively. Conversion to 6 and 6* only proceeded
at 45 °C In CD2Cl2.
A second explanation for the presence of 6a, 6b is to consider
these species as less stable cyclic conformers of 6, 6*. This
lower stability can be attributed to
conformation. This hypothesis is supported by reports of linear
peptides containing N-Me-aa which adopt different
a highly constrained
conformations due to cis/trans interconversion and in some
cases, such as for peptides containing several N-Me-aa, multiple
peaks can be seen by reverse-phase RP-HPLC.[17a, 22] Similar
observations have rarely been reported with cyclotetrapeptides
Scheme 2. Products observed during the synthesis of cyclotetrapeptides (3-7) from their linear precursors (9-13).
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European Journal of Organic Chemistry
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FULL PAPER
As expected, the RP-HPLC trace showed the presence of 9
and 6a as the major species in the reaction mixture and 9* and
6b were observed in lower proportion (supporting information
Figure S30). The HRMS/MS spectra corresponded to that of a
cyclic tetrapeptide (supporting information Figure S31-32).
Remarkably, the 1H NMR spectrum of the reaction mixture at
5 °C, 27 °C and 45 °C did not show traces of the linear peptide 9
(supporting information Figure S33), as evidenced by the
absence of the singlet assigned to the N-methyl protons of the
N-terminal HN-Me-Phe1, which appears upfield (~ 2.42 ppm)
relative to the singlet corresponding to N-methyl protons which
are part of an amide bond (~3.08-2.88 ppm) (Figure 4). This
result indicates that 6a, 6b hydrolysed during the
chromatography, and therefore one can conclude that the RP-
HPLC traces do not show a true picture on the proportions of 9,
protons (~4.88 ppm) (supporting information Figure S34). This is
indicative of a trans configuration for a Leu-N-Me-Phe peptide
bond.[22] The 1D NOE spectra also showed that the same N-
methyl protons (~3.02 ppm) are in slow exchange with another
set of N-methyl protons (~ 2.93 ppm) (supporting information
Figure S34).The 1H NMR spectra of the reaction mixture at
45 °C shows the appearance of three new signals in the N-
methyl protons region that increase in intensity as the reaction
progressed (Figure 5). Two of these signals corresponded with
the chemical shift of the isolated peptides 6 and 6*. Also a
decrease in the intensity of the signal corresponding to 6a and a
smaller signal was observed. The latter was assigned to 6b,
considering that 6a, 6b are the only two cyclic species which are
consumed during the reaction and that are observed by RP-
HPLC before the reaction mixture was heated to 45 °C. NOE
correlations between 6b and the CH protons were not observed
and the CH protons overlapped strongly, making it difficult to
assign correlations among them (supporting information Figure
S35).
1
9* and 6a, 6b in the reaction mixture. The H NMR spectrum of
the crude reaction presents several singlets between ~3.10 and
~2.88 ppm (supporting information Figure S33 and Figure 4),
indicating the presence of N-methyl protons which are part of an
amide bond, thus further confirming a cyclic structure for 6a, 6b.
6
6a
A
B
6*
*
~13.3 hr
~11.0 hr
~
~
~
~
8.2 hr
6.0 hr
3.0 hr
1.0 hr
HNMePhe1
-
-CONMePhe-
6b
3.20
3.15
3.10
3.05
3.00
2.95
2.90
2.85
2.80
2.75
Chemical Shift (ppm)
C
D
Figure 5. 1H NMR spectra (500 MHz, CD2Cl2, 45 °C) of the reaction mixture of
9 (2 mM), T3P (1 equiv.) and DIPEA (2 equiv.) in CD2Cl2 at different time
points. The spectrum shows the N-methyl proton region. * indicates the set of
N-methyl protons which are in exchange with the N-methyl protons of 6a.
-CONMePhe-
In an attempt to elucidate the possible conformations of 6a,6b
we performed density functional theory (DFT) calculations and
molecular dynamics simulations on three different conformers of
6: tctc, tttc and tttt (the cis conformation was assigned to the
Leu-NMePhe amide bonds as established by NMR and the all
cis conformer was omitted from this calculation given the known
poor stability of the cccc conformer in analogous systems).[23]
The calculations were performed in vacuum and in two solvent
systems; DMSO and CH2Cl2, which are available in the
Gaussian 09[27] software (DMSO has similar physico-chemical
properties to DMF). Our calculations showed that in a solvated
system the conformer stability decreases as follows tctc > tttc >
tttt (Table 1). However, there is little energy stabilization
between the tttc and tttt conformers in CH2Cl2. The most stable
conformer is tctc in accord with our experimental results (see
Supporting information Figures S22-23). Additionally, the tttc
conformer displayed a larger dipole moment than tctc and tttt,
which suggests a shorter retention time in reverse phase HPLC
3.5
3.0
2.5
Chemical Shift (ppm)
Figure 4. 1H NMR spectra (500 MHz, CD2Cl2, 27 °C) of A) the reaction mixture
of 9 (2 mM), T3P (1 equiv.) and DIPEA (2 equiv.) in CD2Cl2 after 30 min. at
5 °C, B) linear peptide 9, C) cyclic peptide 6 and D) a mixture of T3P and
DIPEA (1:2) in CD2Cl2.
Furthermore, the 1D NOE NMR spectra showed a weak NOE
correlation between the most intense N-methyl protons observed
in the 1H NMR spectra of the reaction mixture (~3.02 ppm)
(Figure 4A), which based on the RP-HPLC and HRMS/MS
spectra corresponds to 6a, and a signal in the region of the CH
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European Journal of Organic Chemistry
10.1002/ejoc.201601016
FULL PAPER
of the former. The tttt conformer was more stable than the tttc
conformer in vacuum further highlighting the important role of
solvent-peptide interactions on conformer stability. For
comparison purposes, we also performed molecular dynamics
simulations and subsequent energy calculations using standard
molecular mechanics and the GBSA implicit solvent model to
simulate the solvent environment in DMF and CH2Cl2 for the
three conformers of 6. The potential energy (see supporting
information Table S2) calculated for the three conformers
exhibited the same trend (tctc > tttc > tttt) as the DFT
calculations, which is in agreement with our experimental results,
whereby 6a and 6b are kinetically controlled products and 6 the
thermodynamic product. These theoretical calculations
combined with the NMR data indicate that 6a adopts either a tttc
or tttt unstable conformation (Scheme 2), and these are in slow
exchange in solution. Similar conclusions could be drawn after
analyzing the DFT calculations for the three different conformers
of 6*: tctc, tttc and tttt (see supporting information Table S1).
profiles showing the percent of the various products present in
the reaction mixtures for 3-7 at different temperatures are
summarized in Table 2. The data clearly indicates a sequence-
dependent product distribution. Remarkably, during the
synthesis of 7 only the early eluting conformers 7a,7b were
observed at 45 °C. The desired product 7 was obtained in
moderate yields when increasing the temperature to 60 °C
(Table 2), but also an increase in the proportion of 7* was
observed.
Table 2. The percentage of the various products present during the cyclization
of (9-13) to cyclic tetrapeptides (3-7) determined by RP-HPLC-UV peak (210
nm); linear gradient of 5%B-95%B over 45 min (ca. 2%B/min), 1 mL•min-1,
using an XTerra MS C18 column (4.6 mm × 150 mm, 5 µm). A = 0.1% TFA in
H2O and B = 0.1 %TFA in ACN. Reaction conditions: 5 equiv. T3P, 6 equiv.
DIPEA, CH2Cl2/DMF (1:1), 1 day.
3
4
5
6
7
rt 45°C
rt
45°C
rt
45°C
rt
45°C
rt 45°C 60°C
CMa 3% 36% 19% 50% 10% 36% 15% 53%
-
-
3% 39%
4% 22%
Table 1. Optimized structures of the different conformers of 6 in a tube
representation (top). Potential energy differences relative to the tctc conformer
(in kcal/mol) and dipole moments (given in parenthesis in Debye) of the three
minimised conformers of 6 in vacuum, DMSO and CH2Cl2.
ECMb
OCc 28%
-
24% 4% 33%
58%
-
20% 1% 12%
3%
-
-
68%
-
-
52% 40%
-
LPd 36% 23% 13% 10% 16% 17% 47% 17% 42% 36% 18%
ELPe 33% 6%
6% 27% 34% 18% 6% 17% 21%
6%
7%
[a] CM: Cyclic monomer. [b] ECM: epimerised cyclic monomer (3*, 4*, 5*, 6*
and 7*). [c] OC: other conformers (3a,3b, 4a,4b, 5a,5b, 6a,6b and 7a,7b). [d]
LP: linear precursor (10, 11, 12, 9 and 13, respectively). [e] ELP: Epimerised
linear precursor (10*, 11*, 12*, 9* and 13*, respectively).
Finally all the synthetic onychocins reported herein were
tested for their antitumor activity. All the cyclotetrapeptides (3, 4,
5, 6, and 7) moderately inhibit the proliferation of mouse
myeloma cells in a dose-dependent manner, with IC50 values of
13.8 ± 0.2, 45.5 ± 2.5, 23.1 ± 1.7, 13.2 ± 0.2 and 17.2 ± 0.4
µg/mL respectively. Notably, there was a decrease in the
inhibitory activity with a decrease in the size of the side chains of
the amino acids in the cyclotetrapeptide sequence (as
tttt (PEa/μb)
tttc (PE/μ)
tctc (PE/μ)
Vacuum
DMSO
CH2Cl2
8.36/1.00
10.03/4.55
0c/0.21
12.64/1.38
11.74/1.32
11.55/7.19
11.35/6.60
0c/1.33
0c/0.96
exemplified by the IC50 of compounds
4 and 6). This
[a] PE: Potential energy difference (kacl/mol). [b] μ: Dipole moment. [c] The
absolute potential energy for tctc conformer in vacuum, DMSO and CH2Cl2 is -
1108149.20, -1108162.45 and -1108160.10 kcal/mol, respectively.
phenomenon might be indicative of the hydrophobic effect or
potential physical and geometric requirements for molecular
recognition, which could serve as a lead for further drug
development.
With the knowledge gained on the cyclisation of tetrapeptides
using T3P we were then able to prepare the cyclotetrapeptide
analogue 6 of the onychocins (3-5) in acceptable yield while
obtaining a small quantity of the C-terminal epimerized product
(Table 2) by carrying out the cyclisation in the presence of 5
equiv. of T3P, 6 equiv. DIPEA in CH2Cl2/DMF (1:1) for 24 hours
at 45° C using 1 mM peptide concentration. This same protocol
was then used to synthesize the naturally extracted compounds
onychocins (3-5, 7)[6] starting from the corresponding linear
precursors 10-12 and 13, respectively (Table 2). However,
cyclisation yields varied depending on the peptide sequence. To
illustrate this point, the peak area of the chromatographic
Conclusions
The synthesis of the natural onychocins 3-5 and 7 and analogue
6 was successfully accomplished using T3P to effect the key
cyclisation step. Nonetheless, there is a need to further explore
options to restrict carboxylate epimerization to maximise yields.
The cyclisation generates unstable cyclic conformers (kinetic
products) that were detected by RP-HPLC-UV-MS and 1H NMR.
The most stable tctc conformers are favored using longer
reaction times at room temperature or at higher temperatures
This article is protected by copyright. All rights reserved

European Journal of Organic Chemistry
10.1002/ejoc.201601016
FULL PAPER
an Agilent Technologies 1120 Compact LC equipped with a Hewlett-
Packard 1100 MSD mass spectrometer or an Agilent Technologies 1260
Infinity LC equipped with an Agilent Technologies 6120 Quadrupole
mass spectrometer using an analytical column (GraceSmart C18, 120 Å,
4.6 x 150 mm 5 µm) with a 30 min linear gradient of 5-95% solvent B at a
flow rate of 0.3 mL•min-1. Thin layer chromatography (TLC) was
performed using F254 0.2 mm silica plates, followed by visualization with
UV irradiation at 254 nm. Flash column chromatography was performed
using 63−100 μm silica gel.
and their structures agree with the corresponding natural
compounds. Finally, biological evaluation of all the synthetic
cyclotetrapeptides revealed moderate antitumor activity.
Experimental Section
Chemistry
Synthesis of linear peptides
General information: All the reagents purchased from commercial
sources were reagent grade and were used without further purification.
Solvents for peptide synthesis and RP-HPLC were purchased as
synthesis grade and HPLC grade, respectively. 2-Chlorotrityl chloride
resin and ethyl 2-cyano-(hydroxyimino)acetate (Oxyma) were purchased
from Novabiochem (Merck, Germany). O-(Benzotriazol-1-yl)-N,N,N’,N’-
tetramethyluronium hexafluorophosphate (HBTU) and 1-hydroxy-7-
azabenzotriazole (HOAt) were acquired from GL Biochem (Shanghai,
China), while O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium
hexafluorophosphate (HATU) and 6-chloro-1-hydroxybenzotriazole (6-Cl-
HOBt) were purchased from Chempep (Wellington, USA) and Aapptec
(Louisville, USA), respectively. Collidine, pyridine, piperidine, 2-
propanephosphonic acid anhydride (T3P) solution (50% in ethyl acetate),
2,2,2-trifluoroethanol (TFE), N,N-diisopropylethylamine (iPr2NEt) and
N,N’-diisopropylcarbodiimide (DIC) were purchased from Sigma-Aldrich
(St. Louis, USA). N,N-Dimethyl-4-aminopyridine (DMAP) and 1,1,1,3,3,3-
hexafluoropropan-2-ol (HFIP) were purchased from AK Scientific (Union
City, USA). Dimethylformamide (DMF, AR grade), acetonitrile (CH3CN,
HPLC grade) and trifluoroacetic acid (TFA) were purchased from
Scharlau (Barcelona, Spain). Dichloromethane (AR grade) was obtained
from ECP Limited (Auckland, New Zealand). All the Fmoc-amino acids
(Fmoc-Leu-OH, Fmoc-Ile-OH and Fmoc-Val-OH) were purchased from
GL Biochem except Fmoc-N-Me-Phe-OH which was purchased from
Aapptec.
All the linear peptides were prepared on 2-chlorotrityl chloride resin on a
0.1 mmol scale using the Fmoc-SPPS strategy.
H-Leu-NMePhe-Leu-NMePhe-OH (8): A solution of Fmoc-NMePhe-OH
(80 mg, 0.2 mmol) and DIPEA (64.5mg, 0.5 mmol) in CH2Cl2 (3 mL) was
added to the pre-swollen 2-chlorotrityl chloride resin (in CH2Cl2), and the
resulting mixture was agitated at rt for 1 h. The resin was then treated
with a mixture of CH2Cl2:MeOH:DIPEA (16:3:1, 3 mL, 2 × 15 min) in
order to cap the resin unreacted active sites. Fmoc deprotection was
achieved by using 20% piperidine in DMF (5 mL, 2 × 10 min), followed by
amino acid coupling. The two Leu residues were coupled to the
sequence by treating the resin with a solution of Fmoc-Leu-OH (282 mg,
0.8 mmol), HATU (300 mg, 7.9 mmol.) and DIPEA (206 mg, 1.6 mmol) in
5 mL of DMF at rt for 2h and double couplings were performed. The
coupling of N-Me-Phe was accomplished using a mixture of Fmoc-N-
MePhe-OH (160 mg, 0.4 mmol), HATU (148 mg, 3.9 mmol) and DIPEA
(103 mg, 0.8 mmol) in 3 mL of DMF at rt for 45 min. After chain
elongation, the linear peptide was cleaved from the resin by treatment
with HFIP: CH2Cl2 (1:4, 5 mL) at rt for 1h. The resin was filtered and
washed with CH2Cl2 (2 × 1 mL). The resulting filtrates were combined,
followed by removal of solvent and volatile HFIP under a dry nitrogen
flow. Finally the crude reaction mixture was re-dissolved in 50%
acetonitrile aqueous solution, lyophilized and purified by semi-preparative
RP-HPLC to quantitatively afford 8 as a white powder. Anal. RP-HPLC: tR
= 18.28 min (GraceSmart C18, 120 Å, 4.6 x 150 mm, 5 µm, 5% B to 95%
B at 3% B per min, 0.3 mL•min-1). MS (ESI+): C32H46N4O5 [M+H]+
calcd./found 567.3/567.4.
Infrared spectra were recorded on a Perkin Elmer Spectrum 100 infrared
spectrometer, each spectrum was collected with 64 scans at a resolution
of 4 cm-1. Optical rotations were determined at the sodium D line (589
1
nm) at 23 °C using a Perkin Elmer 341 instrument. H, 13C and 1D NOE
nuclear magnetic resonance experiments were performed on a Bruker
AVANCE 400 (1H 400 MHz; 13C 100 MHz) or 500 (1H 500 MHz; 13C 125
MHz) spectrometer in deuterated MeOH, CHCl3, CH2Cl2 or DMSO.
Chemical shifts were recorded in parts per million (ppm) and were
referenced to the residual MeOH signal at 3.31 ppm or the
tetramethylsilane signal at 0.00 ppm. The 13C values were also presented
relative to the signal of residual MeOH or chloroform at 49.0 ppm or 77.0
ppm, respectively. 1H NMR spectral data were reported as follows:
chemical shift (δH), relative integral, multiplicity (s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet; dd, doublet of doublets; dq, doublet of
quartets; ddd, doublet of doublet of doublets) and coupling constant (J in
Hz). The 1D NOE were done with 1 sec mixing time. High-resolution
mass spectra were obtained on a Bruker micrOTOFQ mass spectrometer
using electrospray ionization (ESI) under positive mode. All the analytical
RP-HPLC experiments were carried out using an analytical column
(XTerra MS C18, 125 Å, 4.6 mm × 150 mm, 5 µm) on a Dionex Ultimate
3000 System with a 45 min linear gradient of 5-95% solvent B (where
solvent A was 0.1% TFA in water and solvent B was 0.1% TFA in
acetonitrile) at a flow rate of 1 mL•min-1 and UV signals were detected at
the wavelengths 210, 225, 254 and 280 nm. Semi-preparative RP-HPLC
was carried out on a Waters 600 system using a semi-preparative
column (XTerra MS C18 Prep Column, 125 Å, 19 mm × 300 mm, 10 µm)
at a flow rate of 10 mL•min-1 using a adjusted gradient of 5-95% solvent
B according to the elution profiles obtained from analytical RP-HPLC
chromatography. RP-HPLC-UV-ESIMS analysis was conducted on either
H-NMePhe-Leu-NMePhe-Leu-OH (9): The preparation of
9 was
conducted using a similar procedure to that described above for the
synthesis of 8. Anal. RP-HPLC: tR = 21.2 min (GraceSmart C18, 120 Å,
4.6 x 150 mm, 5 µm, 5% B to 95% B at 3% B per min, 0.3 mL•min-1). MS
(ESI+): C32H46N4O5 [M+H]+ calcd./found 567.3/567.4..
H-NMePhe-Leu-NMePhe-D-Leu-OH (9*): The preparation of 9* was
conducted using a similar procedure to that described above for the
synthesis of 8. Anal. RP-HPLC: tR = 22.53 min (GraceSmart C18, 120 Å,
4.6 x 150 mm, 5 µm, 5% B to 95% B at 3% B per min, 0.3 mL•min-1). MS
(ESI+): C32H46N4O5 [M+H]+ calcd./found 567.3/567.4.
H-NMePhe-Ile-NMePhe-Ile-OH (10): The preparation of 10 was
conducted using a similar procedure to that described above for the
synthesis of 8. Anal. RP-HPLC: tR = 22.9 min (GraceSmart C18, 120 Å,
4.6 x 150 mm, 5 µm, 5% B to 95% B at 3% B per min, 0.3 mL•min-1). MS
(ESI+): C32H46N4O5 [M+H]+ calcd./found 567.3/567.4.
H-NMePhe-Val-NMePhe-Val-OH (11): The preparation of 11 was
performed using a similar procedure to that described above for the
synthesis of 8. Anal. RP-HPLC: tR = 20.1 min (GraceSmart C18, 120 Å,
4.6 x 150 mm, 5 µm, 5% B to 95% B at 3% B per min, 0.3 mL•min-1). MS
(ESI+): C30H42N4O5 [M+H]+ calcd./found 539.3/539.3.
This article is protected by copyright. All rights reserved

European Journal of Organic Chemistry
10.1002/ejoc.201601016
FULL PAPER
H-NMePhe-Ile-NMePhe-Val-OH (12): The preparation of 12 was
performed using a similar procedure to that described above for the
synthesis of 8. Anal. RP-HPLC: tR = 20.8 min (GraceSmart C18, 120 Å,
4.6 x 150 mm, 5 µm, 5% B to 95% B at 3% B per min, 0.3 mL•min-1). MS
(ESI+): C31H44N4O5 [M+H]+ calcd./found 553.2/553.4.
J=8.1, 2 H, 2 × α-CH (Val)), 3.69 (d, J=12.1, 2 H, 2 × β-CH2a (NMePhe)),
3.03 (dd, J=15.1, 11.3, 2 H, 2 × β-CH2b (NMePhe)), 2.87 (s, 6 H, 2 × N-
CH3), 2.13 (dd, J=14.1, 6.9, 2 H, 2 × β-CH (Val)), 0.89 (d, J=6.8, 6 H, 2 ×
CHCH3), 0.74 (d, J=6.4, 6 H, 2 × CHCH3). 13C NMR (100 MHz, MeOD): δ
= 173.3 (C=O (Val)), 172.1 (C=O (NMePhe)), 138.7 (CAr), 130.0 (CHAr),
129.5 (CHAr), 128.1 (CHAr), 64.6 (α-CH (NMePhe)), 57.3 (α-CH (Val)),
35.2 (β-CH2 (NMePhe)), 31.2 (N-CH3), 20.9 (CHCH3), 18.5 (CHCH3)
H-NMePhe-Leu-NMePhe-Val-OH (13): The preparation of 13 was
performed using a similar procedure to that described above for the
synthesis of 8. Anal. RP-HPLC: tR = 22.0 min (GraceSmart C18, 120 Å,
4.6 x 150 mm, 5 µm, 5% B to 95% B at 3% B per min, 0.3 mL•min-1). MS
(ESI+): C31H44N4O5 [M+H]+ calcd./found 553.2/553.4.
ppm;
HRMS
(ESI+):
C30H40N4O4Na
[M+Na]+
calcd./found
543.3050/543.2951.
Cyclo[Ile-NMePhe-Val-NMePhe] (5): 5 was obtained as a colorless
powder (6.1 mg, 36%) from its linear precursor 12 (18.1 mg, 32.8 μmol)
using a similar method to that described for 6. Anal. RP-HPLC: tR = 30.2
Synthesis of cyclotetrapeptides
min (XTerra MS C18, 125 Å, 4.6 mm × 150 mm, 5 µm, 5% B to 95% B at
23.2
2% B per min, 1 mL•min-1); [α]D
-149.23 (c 0.1, CH2Cl2); IR (cm-1):
Cyclo[Leu-NMePhe-Leu-NMePhe] (6): To a solution of 9 (17.1 mg, 30.2
μmol) and T3P (90 μL, 151.0 μmol) in CH2Cl2:DMF (1:1, 30 mL) was
added DIPEA (31.6 μL, 181.2 μmol) and the reaction mixture was stirred
at 45 °C for 1 day. After evaporation of CH2Cl2 under reduced pressure,
the reaction residue was purified by semi-preparative RP-HPLC to give 6
as a colorless powder (8.8 mg, 53%). Anal. RP-HPLC: tR = 31.6 min
(XTerra MS C18, 125 Å, 4.6 mm × 150 mm, 5 µm, 5% B to 95% B at 2%
B per min, 1 mL•min-1); [α]D23.2 -218.75 (c 0.08, CH2Cl2); IR (cm-1): 3317,
3064, 3027, 2956, 2927, 2870, 1655, 1629 1498, 1454, 1402, 1323,
1300, 1172, 1090, 965, 731, 700. 1H NMR (400 MHz, CDCl3): δ = 7.31 (t,
J=7.4, 4 H, CHAr), 7.24 (d, J=7.2, 6 H, CHAr), 6.99 (d, J=9.2, 2 H, 2 ×
CONH), 4.61 (m, 4 H, 2 × α-CH (Leu) and 2 × α-CH (NMePhe)), 3.77 (d,
J=13.9, 2 H, 2 × β-CH2a (NMePhe)), 2.89 (d, J=12.0, 2 H, 2 × β-CH2b
(NMePhe)), 2.83 (s, 6 H, 2 × N-CH3), 1.66 (m, 2 H, 2 × β-CH2a (Leu) ),
1.40 (m, 2 H, 2 × γ-CH (Leu) ), 1.29 (d, J=6.0, 2 H, 2 × β-CH2b (Leu)),
0.84 (d, J=6.5, 6 H, 2 × CHCH3), 0.78 (d, J=6.6, 6 H, CHCH3) ppm. 13C
NMR (100 MHz, CDCl3): δ = 173.4 (C=O (NMePhe)), 170.1 (C=O (Leu)),
137.4 (CAr), 129.6 (CHAr), 128.6 (CHAr), 127.7 (CHAr), 63.8 (α-CH
(NMePhe)), 49.2 (α-CH (Leu)), 41.4 (β-CH2 (Leu)), 34.9 (β-CH2
(NMePhe)), 31.0 (N-CH3), 25.2 (γ-CH (Leu)), 22.9 (CHCH3), 22.8
(CHCH3) ppm; HRMS (ESI+): C32H44N4O4Na [M+Na]+ calcd./found
571.3255/571.3259.
3323, 3065, 3031, 2967, 2933, 2877, 1657, 1630, 1498, 1455, 1404,
1386, 1320, 1169, 1089, 975, 737, 700; H NMR (400 MHz, MeOD) δ =
1
7.31 (m, 10 H, CHAr), 4.50 (m, 2 H, 2 × α-CH (NMePhe)), 4.31 (t, J=7.5, 1
H, α-CH (Ile)), 4.25 (t, J=7.5, 1 H, α-CH (Val)), 3.63 (d, 14.9, 2 H, 2 × β-
CH2a (NMePhe)), 3.02 (dd, J=14.9, 11.5, 2 H, 2 × β-CH2b (NMePhe)),
2.87 (s, 3 H, N-CH3), 2.86 (s, 3 H, N-CH3), 2.12 (dd, J=14.0, 7.0, 1 H, β-
CH (Val)), 1.86 (m, 1 H, β-CH (Ile)), 1.50 (m, 1 H, γ-CH2a (Ile)), 1.01 (m, 1
H, γ-CH2b (Ile)), 0.89 (t, J=6.4, 6 H, CH2CH3 (Ile) and CHCH3 (Val)), 0.72
(dd, J=12.4, 6.4, 6 H, CHCH3 (Val) and CHCH3 (Ile) ) ppm. 13C NMR (125
MHz, MeOD): δ = 173.6 (C=O (Val) and C=O (Ile) ), 172.1 (C=O
(NMePhe)), 139.0 (CAr), 130.1 (CHAr), 129.7 (CHAr), 128.3 (CHAr), 64.7
(α-CH (NMePhe)), 57.5 (α-CH (Val)), 56.7 (α-CH (Ile)), 36.9 (β-CH2 (Ile)),
35.3 (β-CH2 (NMePhe)), 31.4 (N-CH3), 30.4 (β-CH (Val)), 25.8 (γ-
CH2(Ile)), 20.9 (CHCH3 (Val)), 18.7 (CHCH3 (Val)), 17.2 (CHCH3 (Ile)),
12.1 (CH2CH3) ppm; HRMS (ESI+): C31H42N4O4Na [M+Na]+ calcd./found
557.3206/557.3089.
Cyclo[Leu-NMePhe-Val-NMePhe] (7): 7 was obtained as a colorless
powder (8.8 mg, 40%) from its linear precursor 13 (22.7 mg, 41.1 μmol)
using a similar method to that described for 6 but the reaction was
carried out at 60 °C. Anal. RP-HPLC: tR = 30.4 min (XTerra MS C18, 125
Å, 4.6 mm × 150 mm, 5 µm, 5% B to 95% B at 2% B per min, 1 mL•min-
23.2
1); [α]D
-173.00 (c 0.2, CH2Cl2); IR (cm-1): 3323, 3063, 3031, 2961,
Cyclo[Ile-NMePhe-Ile-NMePhe] (3): 3 was obtained as a colorless
powder (7.7 mg, 36%) from its linear precursor 10 (22.1 mg, 39.0 μmol)
using a similar method to that described for 6. Anal. RP-HPLC: tR = 31.6
2928, 2872, 1654, 1626, 1498, 1455, 1403, 1387, 1322, 1167, 1089, 975,
731, 700; 1H NMR (400 MHz, CDCl3) δ = 7.23 (m, 10 H, CHAr), 6.89 (t, 8.5,
2 H, CONH (Leu) and CONH (Val)), 4.59 (m, 3 H, 2 × α-CH (NMePhe) and
α-CH (Leu)), 4.23 (t, 8.9, 1 H α-CH (Val)), 3.77 (t, 13.6, 2 H, 2 × β-CH2a
(NMePhe)), 2.90 (m, 2 H, 2 × β-CH2b (NMePhe)), 2.85 (s, 3 H, N-CH3),
2.81 (s, 3 H, N-CH3), 2.08 (m, 1 H, β-CH (Val)), 1.66 (m, 2 H, 2 × β-CH2a
(Leu) ), 1.39 (m, 2 H, 2 × γ-CH (Leu) ), 1.25 (m, 2 H, 2 × β-CH2b (Leu)),
0.84 (t, J=6.7, 3 H, CHCH3), 0.77 (d, J=6.6, 3 H, CHCH3) ppm.13C NMR
(100 MHz, CDCl3): δ = 173.2 C=O (Val)), 172.5 (C=O (NMePhe)), 169.8
(C=O (Leu)), 137.1 (CAr), 129.2 (CHAr), 128.4 (CHAr), 127.4 (CHAr), 63.6
(α-CH (NMePhe)), 63.2 (α-CH (NMePhe)), 56.2 (α-CH (Val)), 49.0 (α-CH
(Leu)), 41.0 (β-CH2 (Leu)), 34.8 (β-CH2 (NMePhe)), 31.0 (N-CH3), 29.3
(β-CH (Val)), 24.7 (γ-CH(Leu)), 22.7 (CHCH3 (Leu)), 22.5 (CHCH3 (Leu)),
22.2 (CHCH3 (Val)), 18.3 (CHCH3 (Val)) ppm; HRMS (ESI+):
C31H42N4O4Na [M+Na]+ calcd./found 557.3206/557.3206.
min (XTerra MS C18, 125 Å, 4.6 mm × 150 mm, 5 µm, 5% B to 95% B at
23.2
2% B per min, 1 mL•min-1); [α]D
-153.00 (c 0.1, CH2Cl2); IR (cm-1):
3328, 3065, 3032, 2967, 2933, 2877, 1652, 1628, 1498, 1455, 1405,
1321, 1298, 1168, 1089, 973, 738, 700;δ = 1H NMR (400 MHz, MeOD):
1H NMR (400 MHz, MeOD): δ = 7.32 (m, 10 H, CHAr), 4.51 (m, 2 H, 2 ×
α-CH (Ile)), 4.32 (t, J=8.7, 2 H, 2 × α-CH (NMePhe)), 3.69 (d, J=13.3, 2 H,
2 × β-CH2a (NMePhe)), 3.03 (m, 2 H, 2 × β-CH2b (NMePhe)), 2.88 (s, 6 H,
2 × N-CH3), 1.87 (m, 2 H, 2 × β-CH (Ile)), 1.52 (dd, J=12.7, 7.7, 2 H, 2 ×
γ-CH2a (Ile)), 1.02 (dt, J=16.2, 6.9, 2 H, 2 × γ-CH2b (Ile)), 0.90 (t, J=7.3, 6
H, 2 × CH2CH3), 0.72 (d, J=6.3, 6 H, 2 × CHCH3) ppm. 13C NMR (100
MHz, MeOD): δ = 173.5 (C=O (Ile)), 172.2 (C=O (NMePhe)), 138.8 (CAr),
130.1 (CHAr), 129.7 (CHAr), 128.3 (CHAr), 64.6 (α-CH (NMePhe)), 56.8 (α-
CH (Ile)), 37.0 (β-CH (Ile)), 35.1 (β-CH2 (NMePhe)), 31.5 (N-CH3), 25.6
(γ-CH2 (Ile)), 17.2 (CHCH3 ), 12.0 (CH2CH3) ppm; HRMS (ESI+):
C32H45N4O4 [M+H]+ calcd./found 549.3363/549.3425.
Molecular modeling
The structures of three different conformers were built in Discovery
Studio 3.0 (DS) using the Charmm Forcefield.[28] The geometry
optimizations and energy calculations of the resulting structures were
then carried out in Gaussian 09 at the B3LYP/6-31G(d,p) level with
standard settings. Solvation in CH2Cl2 and DMSO was modelled by a
polarizable continuum model. For comparison, the three backbone
conformers were also submitted to a constrained molecular dynamics
simulation at a time step of 1 fs. The constraints included only the
Cyclo[Val-NMePhe-Val-NMePhe] (4): 4 was obtained as a colorless
powder (12.2 mg, 50%) from its linear precursor 11 (25.3 mg, 47.0 μmol)
using a similar method to that described for 6. Anal. RP-HPLC: tR = 28.9
min (XTerra MS C18, 125 Å, 4.6 mm × 150 mm, 5 µm, 5% B to 95% B at
23.2
2% B per min, 1 mL•min-1); [α]D
-205.25 (c 0.08, CH2Cl2); IR (cm-1):
3323, 3065, 3031, 2966, 2931, 2875, 1662, 1629, 1497, 1454, 1404,
1386, 1172, 1089, 978, 733, 699; 1H NMR (400 MHz, MeOD) δ = 7.32 (m,
10 H, CHAr), 4.50 (dd, J=11.5, 3.0, 2 H, 2 × α-CH (NMePhe)), 4.24 (d,
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European Journal of Organic Chemistry
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dihedral angle of the two methylated amide bonds with values set as 0°
or 180° referring to cis and trans conformations respectively, and the
constant force was set to 0.05 kcal/mol•deg. The detailed procedures of
molecular dynamics are summarized as follows: Minimised structure was
heated to 300 K for 20 ps, followed by 100 ps equilibration at the same
temperature. The production phase was performed for 10 ns with
samples taken at an interval of 1 ps. The final energy minimisation of the
resulting conformations from MD was achieved by 1000 steps of steepest
descent followed by another 500 steps of conjugate gradient
minimisation. The GBSA implicit solvent model using the dielectric
constant of DMF (38) and CH2Cl2 (9) was applied for simulations in DMF
and CH2Cl2.
Hutt, C. A. Olsen, C. J. Vickers, D. Herman, M. A. Chalfant, A.
Montero, L. J. Leman, R. Burkle, B. E. Maryanoff, W. E. Balch, M.
R. Ghadiri, ACS Med. Chem. Lett. 2011, 2, 703-707; c) M. P. I.
Bhuiyan, T. Kato, T. Okauchi, N. Nishino, S. Maeda, T. G. Nishino,
M. Yoshida, Bioorg. Med. Chem. 2006, 14, 3438-3446; dS. Mei, A.
D. Ho, U. Mahlknecht, Int. J. Oncol. 2004, 25, 1509-1519.
Y. Terui, Y. w. Chu, J. y. Li, T. Ando, T. Fukunaga, T. Aoki, Y.
Toda, Bioorg. Med. Chem. Lett. 2008, 18, 6321-6323.
I. Pérez-Victoria, J. Martín, V. González-Menéndez, N. de Pedro,
N. El Aouad, F. J. Ortiz-López, J. R. Tormo, G. Platas, F. Vicente,
G. F. Bills, O. Genilloud, M. A. Goetz, F. Reyes, J. Nat. Prod. 2012,
75, 1210-1214.
[5]
[6]
Bioassays
[7]
W. L. Meyer, G. E. Templeton, C. I. Grable, C. W. Sigel, R. Jones,
S. H. Woodhead, C. Sauer, Tetrahedron Lett. 1971, 12, 2357-2360.
G. Lang, J. W. Blunt, N. J. Cummings, A. L. J. Cole, M. H. G.
Munro, J. Nat. Prod. 2005, 68, 1303-1305.
The peptides were dissolved in DMSO to provide 10 mg/mL stock
solutions. An aliquot of each stock solution was transferred to the first
lane of Rows B to G in a 96-well microtitre plate and two-fold serially
diluted across the 12 lanes of the plate to provide a 2,048-fold
concentration gradient. Bioassay medium was added to an aliquot of
each test solution to provide a 100-fold dilution into the final bioassay,
thus yielding a test range of 100 to 0.05 g/mL. Row A was used as the
positive control (no inhibition) and Row H was used as the negative
control (complete inhibition). CyTOX is an indicative bioassay platform for
discovery of antitumour actives. NS-1 (ATCC TIB-18) mouse myeloma
cells were inoculated in 96-well microtitre plates (190 L) at 50,000
cells/mL in DMEM (Dulbecco's Modified Eagle Medium + 10% fetal
bovine serum (FBS) + 1% penicillin/streptomycin (Life Technologies))
and incubated in 37 °C (5% CO2) incubator. At 48 h, resazurin (120
g/mL; 10 L) was added to each well and the plates were incubated for a
further 48 h. Finally, the absorbance of each well at 605 nm was
measured using a Spectromax plate reader (Molecular Devices).
[8]
[9]
C. Almeida, F. El Maddah, S. Kehraus, G. Schnakenburg, G. M.
König, Org. Lett. 2016, 18, 528-531.
[10]
H. Guo, N. B. Kreuzenbeck, S. Otani, M. Garcia-Altares, H. M.
Dahse, C. Weigel, D. K. Aanen, C. Hertweck, M. Poulsen, C.
Beemelmanns, Org. Lett. 2016, 18, 3338-3341.
[11]
[12]
H. Kawagishi, A. Somoto, J. Kuranari, A. Kimura, S. Chiba,
Tetrahedron Lett. 1993, 34, 3439-3440.
a) D. A. Horton, G. T. Bourne, J. Coughlan, S. M. Kaiser, C. M.
Jacobs, A. Jones, A. Ruhmann, J. Y. Turner, M. L. Smythe,
Organic & Biomolecular Chemistry 2008, 6, 1386-1395; b) K. A.
Fairweather, N. Sayyadi, I. J. Luck, J. K. Clegg, K. A. Jolliffe, Org.
Lett. 2010, 12, 3136-3139.
[13]
[14]
L. M. De Leon Rodriguez, A. J. Weidkamp, M. A. Brimble, Org.
Biomol. Chem. 2015, 13, 6906-6921.
Acknowledgements
a) W. Mastle, U. Link, W. Witschel, U. Thewalt, T. Weber, M.
Rothe, Biopolymers 1991, 31, 735-744; b) J. Pastuszak, J. H.
Gardner, J. Singh, D. H. Rich, J. Org. Chem. 1982, 47, 2982-2987.
C. A. Lipinski, F. Lombardo, B. W. Dominy, P. J. Feeney, Adv.
Drug Deliv. Rev. 1997, 23, 3-25.
We sincerely thank Mr Daniel Vuong (MST) for performing the
bioassay and Mr. Yi Zou from China Pharmaceutical University
for providing access to the Gaussian 09 and Discovery Studio
3.0 software packages. The authors would also like to thank the
China Scholarship Council (S.Z.) and the Australian Research
Council (FT130100142; A.P.) for financial support.
[15]
[16]
[17]
M. J. O. Anteunis, C. Van Der Auwera, Int. J. Pept. Prot. Res.
1988, 31, 301-310.
aM. Teixidó, F. Albericio, E. Giralt, J. Pept. Res. 2005, 65, 153-
166; bR. Roodbeen, S. L. Pedersen, M. Hosseini, K. J. Jensen,
Eur. J. Org. Chem. 2012, 7106-7111.
Keywords: cyclisation • tetrapeptides • conformational analysis•
natural products • biological activity
[18]
[19]
a) K. Titlestad, Acta Chem Scand B 1977, 31, 523-526; b) S. A.
Miller, S. L. Griffiths, D. Seebach, Helv. Chim. Acta 1993, 76, 563-
595; c) D. Seebach, O. Bezençon, B. Jaun, T. Pietzonka, J. L.
Matthews, F. N. M. Kühnle, W. B. Schweizer, Helv. Chim. Acta
1996, 79, 588-608.
[1]
[2]
[3]
W. L. Meyer, L. F. Kuyper, D. W. Phelps, A. W. Cordes, J. Chem.
Soc. Chem. Comm. 1974, 339-340.
E. L. Kim, J. L. Li, B. Xiao, J. Hong, E. S. Yoo, W. D. Yoon, J. H.
Jung, Chem. Pharm. Bull. 2012, 60, 1590-1593.
a) H. A. Lim, C. Kang, C. S. B. Chia, Int. J. Pept. Res. Ther. 2010,
16, 145-152; b) L. L. Ling, T. Schneider, A. J. Peoples, A. L.
Spoering, I. Engels, B. P. Conlon, A. Mueller, T. F. Schäberle, D. E.
Hughes, S. Epstein, M. Jones, L. Lazarides, V. A. Steadman, D. R.
Cohen, C. R. Felix, K. A. Fetterman, W. P. Millett, A. G. Nitti, A. M.
Zullo, C. Chen, K. Lewis, Nature 2015, 517, 455-459.
a) D. H. Rich, P. Mathiaparanam, Tetrahedron Lett. 1974, 15,
4037-4040; b) D. H. Rich, P. Bhatnagar, P. Mathiaparanam, J. A.
Grant, J. P. Tam, J. Org. Chem. 1978, 43, 296-302; c) J. V.
Edwards, A. R. Lax, E. B. Lillehoj, G. J. Boudreaux, Int. J. Pept.
Prot.Res. 1986, 28, 603-612; d) F. Cavelier, J. Verducci,
Tetrahedron Lett. 1995, 36, 4425-4428; e) N. Loiseau, F. Cavelier,
J. P. Noel, J. M. Gomis, J. Pept. Sci. 2002, 8, 335-346.
R. Dahiya, M. Maheshwari, A. Kumar, Monatsh. Chem. 2009, 140,
121-127.
[4]
a) Y. Nakao, S. Yoshida, S. Matsunaga, N. Shindoh, Y. Terada, K.
Nagai, J. K. Yamashita, A. Ganesan, R. W. M. Van Soest, N.
Fusetani, Angew. Chem. Int. Ed. 2006, 45, 7553-7557; b) D. M.
[20]
This article is protected by copyright. All rights reserved

European Journal of Organic Chemistry
10.1002/ejoc.201601016
FULL PAPER
[21]
aJ. Klose, M. Bienert, C. Mollenkopf, D. Wehle, C. W. Zhang, L. A.
Carpino, P. Henklein, Chem. Commun. 1999, 1847-1848; bJ. R.
Dunetz, Y. Xiang, A. Baldwin, J. Ringling, Org. Lett. 2011, 13,
5048-5051.
[22]
D. Sýkora, L. Žáková, M. Buděšínský, J. Chromatogr. A 2007,
1160, 128-136.
[23]
[24]
[25]
[26]
Y. Che, G. R. Marshall, J. Med. Chem. 2006, 49, 111-124.
X. Yan, R. M. Bain, R. G. Cooks, Angew. Chem. Int. Ed. 2016.
E. Valeur, M. Bradley, Chem. Soc. Rev. 2009, 38, 606-631.
D. H. Rich, P. K. Bhatnagar, J. Am. Chem. Soc. 1978, 100, 2218-
2224.
[27]
[28]
Gaussian 09, Revision B.09, Gaussian Inc., Wallingford CT, 2009.
B. R. Brooks, R. E. Bruccoleri, B. D. Olafson, D. J. States, S.
Swaminathan, M. Karplus, J. Comput. Chem. 1983, 4, 187-217.
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The syntheses of a series of
Cyclotetrapeptides synthesis
naturally
cyclotetrapeptides
analogue
propanephosphonic
anhydride is reported. An
unexpected finding is the
formation of unstable cyclic
peptide conformers in addition
to the trans,cis,trans,cis most
stable cyclopeptides.
occurring
Shengping Zhang, Luis M. De
Leon Rodriguez, Ernest Lacey,
Andrew M. Piggott and Margaret
A. Brimble*
and
an
2-
with
acid
Page No. – Page No.
Cyclisation
of
Linear
Tetrapeptides Containing N-
Methylated Amino Acids Using
2-Propanephosphonic
Anhydride
Acid
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