Novel method for the synthesis of urea backbone cyclic peptides using new Alloc-protected glycine building units

Article abstract of DOI:10.1002/psc.1218

Cyclization of bioactive peptides, utilizing functional groups serving as natural pharmacophors, is often accompanied with loss of activity. The backbone cyclization approach was developed to overcome this limitation and enhance pharmacological properties. Backbone cyclic peptides are prepared by the incorporation of special building units, capable of forming amide, disulfide and coordinative bonds. Urea bridge is often used for the preparation of cyclic peptides by connecting two amine functionalized side chains.Herewe present urea backbone cyclization as an additional method for the preparation of backbone cyclic peptide libraries. A straightforward method for the synthesis of crystalline Fmoc-N^α [ω-amino(Alloc)-alkyl] glycine building units is presented. A set of urea backbone cyclic Glycogen Synthase Kinase 3 analogs was prepared and assessed for protein kinase B inhibition as anticancer leads. Copyright

Full text of DOI:10.1002/psc.1218

Research Article
Received: 8 November 2009
Revised: 11 January 2010
Accepted: 15 January 2010
Published online in Wiley Interscience: 1 March 2010
(
www.interscience.com) DOI 10.1002/psc.1218
Novel method for the synthesis of urea
backbone cyclic peptides using new
Alloc-protected glycine building units
a‡
a‡
b
a
Mattan Hurevich, Yftah Tal-Gan, Shoshana Klein, Yaniv Barda,
b
a∗
Alexander Levitzki and Chaim Gilon
Cyclization of bioactive peptides, utilizing functional groups serving as natural pharmacophors, is often accompanied with
loss of activity. The backbone cyclization approach was developed to overcome this limitation and enhance pharmacological
properties. Backbone cyclic peptides are prepared by the incorporation of special building units, capable of forming amide,
disulfide and coordinative bonds. Urea bridge is often used for the preparation of cyclic peptides by connecting two amine
functionalized side chains. Here we present urea backbone cyclization as an additional method for the preparation of backbone
α
cyclic peptide libraries. A straightforward method for the synthesis of crystalline Fmoc-N [ω-amino(Alloc)-alkyl] glycine
building units is presented. A set of urea backbone cyclic Glycogen Synthase Kinase 3 analogs was prepared and assessed for
protein kinase B inhibition as anticancer leads. Copyright ꢀc 2010 European Peptide Society and John Wiley & Sons, Ltd.
Keywords: urea backbone cyclization; solid phase peptide synthesis; cyclic peptides; AGBU
Introduction
of these chemical entities on the bridge. Previous studies have
shown that the chemical bond used for cyclization can affect
the conformation and bioactivity of backbone cyclic peptides
Linear peptides have a number of drawbacks as drug candidates,
mainly the lack of important pharmacological properties such
as metabolic stability and oral bioavailability. Many local and
global modifications have been developed to improve the
pharmacological properties of active peptides. Cyclization is
one of the most common strategies used to prepare peptide-
based drugs. Cyclization of natural bioactive peptides is not
always feasible, either due to lack of suitable side chain
functionalities for bond formation or because the functional
groupsarecrucialforthebioactivity[1,2].Thebackbonecyclization
methodology [3] overcomes these limitations by conferring long-
range conformational constraint upon the peptide backbone with
minimal alterations to the native peptide sequence. Ring closure
is achieved by artificial, functionalized alkyl spacers anchored to
the peptide backbone that can either be covalently connected to
another appropriate functionalized spacer, to a functional amino
acid side chain on the peptide or to the N- or C-terminus of the
peptide. Cyclization can thus be accomplished without changing
the original sequence required for bioactivity. Restricting the
conformational space of peptide structures by cyclization usually
results in inactive peptides; thus, libraries should be screened
to increase the probability of finding a backbone cyclic peptide
that overlaps with the bioactive conformation of the original
peptide [4–7]. Active backbone cyclic peptides discovered by
libraryscreeninghavebeenproventopossessenhancedselectivity
[
12,13]. Extensive work has been dedicated to establishing new
methods for the bridge bond formation of cyclic peptides. Novel
approaches for bond formation such as metal cyclization [14,15]
and metathesis mediated cyclization [16,17], in addition to amide
[18] and disulfide bridge [19–24] have been used to synthesize
new classes of biopharmaceutical cyclic peptides. Many of these
methods have been adapted for the preparation of biologically
relevantbackbonecyclicpeptides,e.g.amide[9],Azo[25],disulfide
[8], metathesis [13,26,27] and metal ion complexation [25].
Urea bridge formation [28,29] was reported for the preparation
of cyclic analogs of Enkephalin in solution and in SPPS [30,31]
utilizing amino side chains (e.g. lysine, ornithine). In addition,
we recently introduced a new procedure for the synthesis of
urea macrocycles on a solid support [32]. This procedure enables
straightforward formation of cyclic urea from two free amines
anchored to the resin. Here we present urea backbone cyclization
∗
Correspondence to: Chaim Gilon, Institute of Chemistry, The Hebrew University
of Jerusalem, 91904 Jerusalem, Israel. E-mail: gilon@vms.huji.ac.il
a Institute of Chemistry, The Hebrew University of Jerusalem, 91904 Jerusalem,
Israel
b Unit of Cellular Signaling, Department of Biological Chemistry, The Alexander
Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, 91904
Jerusalem, Israel
[8], potency [9], bioavailability and metabolic stability compared
to linear peptides [10,11].
Conformational libraries of backbone cyclic peptides can be
prepared by varying four parameters: the mode of cyclization,
ring position, ring size and ring chemistry. Ring chemistry is
defined by the type of chemical functions that exist on the bridge,
the chemistry of the bond used for cyclization and the position
‡
These authors contributed equally to this work.
Abbreviations used: EGTA, ethylene glycol tetraacetic acid; LTQ, linear trap
quadrupole; IUPAC-IUB, International Union of Pure and Applied Chemistry-
International Union of Biochemistry.
J. Pept. Sci. 2010; 16: 178–185
Copyright ꢀc 2010 European Peptide Society and John Wiley & Sons, Ltd.

NOVEL METHOD FOR THE SYNTHESIS OF UREA BACKBONE CYCLIC PEPTIDES
as a new bond formation method for the preparation of backbone
cyclic peptides. In this method, the urea bridge is formed between
two N-alkylated glycine derivatives bearing an amino moiety on
the alkyl chain. The urea backbone cyclization approach is an
alternative to standard amide backbone cyclization. It can be used
to increase the diversity of backbone cyclic peptides by alteration
of the bridge chemistry and the position of the urea bond in the
bridge.
Improved procedures for the large-scale preparation of
Fmoc-[N-(Alloc)ω-aminoalkyl]glycine building units [called herein
alloc glycine buiding units (AGBU)] and their incorporation into
peptide sequence using standard Fmoc SPPS protocols are also
described.
Persistently activated protein kinase B (PKB/Akt) is associated
with many human cancers, such as breast, colon, ovary, pancreas,
head and neck, and prostate [33]. Therefore, PKB/Akt is considered
an attractive candidate for targeted cancer therapy. The use
of substrate mimetic peptides is an appealing strategy for the
inhibition of protein kinases [34,35]. Recently, a series of peptides
derived from the PKB/Akt substrate protein Glycogen Synthase
Kinase 3 (GSK3) were synthesized and their interaction with
PKB was studied [36], leading to the discovery of a selective
PKB/Akt inhibitor, PTR6154 (Arg-Pro-Arg-Nva-Tyr-Dap-Hol). The
urea backbone cyclization method was applied to the preparation
of four cyclic peptides based on PTR6154. The compounds were
screened by evaluating the in vitro inhibitory effect of the cyclic
peptides on PKB/Akt function.
Mono Alloc-protected diamines 1a–d were obtained by
treating allylchloroformate (Alloc-Cl) with a tenfold excess of
the corresponding diamine (Scheme 1). Reductive alkylation of
mono Alloc-protected diamines 1a–d was performed by the
simultaneous addition of 0.85 molar equivalent of NaCNBH3 and
α
glyoxylic acid to yield 2a–d. The unprotected N [ω-amino(Alloc)-
alkyl] glycine 2a–d were not isolated, and the reaction mixture
was treated with 0.75 molar equivalent of Fmoc-OSu to produce
AGBU 3a–d. We found that using sub-equivalent amounts of
reagents (glyoxylic acid and Fmoc-OSu) significantly increased
product purity and avoided tedious purification workup. The final
products, 3a–d, were obtained as sticky colorless pastes [over
0%purityasdeterminedbyhighpressureliquidchromatography
HPLC)].HandlingoftheseoilyAGBUwasinconvenientforstandard
peptide synthesis.
Crystallization protocols were developed to obtain 3a–d as
granular powders, in an attempt to increase the yield and purity
of backbone cyclic peptides. Crude 3a–c were recrystallized
from boiling toluene, yielding sticky white pellets that gave
white powder after extensive washing with petroleum ether.
Crystallization of 3d was achieved by slow addition of hexane to
a toluene solution at room temperature. These protocols yielded
8
(
3
a–d as pure compounds (over 95% purity as determined by
HPLC) that proved convenient for handling and storage. This
procedure presents a general method for large-scale synthesis of
amino-alkylated glycine derivatives using commercially available
starting materials.
Todemonstratetheusefulnessofthisprocedure,wesynthesized
four backbone cyclic peptides by standard Fmoc SPPS protocols
using the crystallized AGBU.
Results and Discussion
Design and Synthesis of the YM Analogs
Synthesis of Alloc Glycine Building Units
In amide backbone cyclization, the cyclization can be performed
either by connecting a dicarboxylic linker to the amino terminus
of the peptide [9,10,12,25], which prevents peptide elongation
beyond the point of cyclization, or by the use of an additional
set of allyl ω-carboxy building units, appropriate for Fmoc SPPS
protocols [44]. Urea backbone cyclization can overcome these
limitations by incorporating two AGBU in the peptide sequence.
In order to mimic the inhibitory effect of PTR6154, four urea
backbone cyclic analogs of c(YM3-m) were designed. The proline
residue (cyclic N-alkylated amino acid) in PTR6154 was substituted
byanAGBU(N-alkylatedglycinemoiety)thatallowedbothpeptide
elongation and cyclization. An additional AGBU was introduced at
thecarboxyterminus.Allfourcompoundshavethesamesequence
but differ in the bridge size. Previous studies indicated that the
amino terminus plays a crucial role in PKB/Akt inhibition (Tal-Gan
et al., in preparation), thus the amino terminus was protected
throughout the cyclization step and the Fmoc protecting group
was removed only in the final step of the peptide synthesis, before
cleavage of the peptides from the resin.
α
N (ω-functionalizedalkylated)glycinederivativesbearingorthog-
onal protected amino moieties are used as building blocks for the
synthesisofbackbonecyclicpeptides[12],peptoids[37–39],pseu-
dopeptides[40], andinthesynthesisofpeptidenucleicacids(PNA)
α
[
41,42]. Glycine derivatives of the type Fmoc-N [ω-amino(Boc)-
alkyl] glycine are commercially available but cannot be used for
the synthesis of backbone cyclic peptides using standard SPPS
protocols.
The allyloxycarbonyl group (Alloc) has been widely used for the
protectionofprimaryamines[43]andhasproventobeorthogonal
to the removal conditions of both Fmoc and Boc groups. We report
a procedure for gram scale synthesis of a variety of AGBU, suitable
for the preparation of backbone cyclic peptides using standard
Fmoc SPPS.
AGBU (3a–d) (Scheme 1) were synthesized in three steps. The
procedure was based on mono Alloc protection of diamines,
followed by reductive alkylation in the presence of glyoxylic acid
and in situ Fmoc protection.
Alloc
Alloc
NH
n
NH
O
C
O
C
i
ii
iii
n
Alloc
NH2
n NH2
H N
n N
H
HN
N
2
OH
OH
CH2
Fmoc
CH2
a, n=2
b, n=3
c, n=4
d, n=6
1a-d
3a-d
Scheme 1. Synthesis of AGBU. Conditions: (i) Allylchloroformate; (ii) Glyoxylic acid, NaCNBH3; (iii) Fmoc-OSu, Et3N.
J. Pept. Sci. 2010; 16: 178–185 Copyright ꢀc 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

HUREVICH ET AL.
Alloc NH
Alloc NH
(CH2)3
Alloc NH
Alloc NH
(CH2)3
(
CH2)3
(CH )m
2
a,b,c,a
d
a,b
Fmoc
NH CH2 CO
Fmoc Hol
N
CH2 CO
Fmoc N CH2
C
O
Arg Nva Tyr Dap Hol
Pbf tBu Boc
N
CH2 CO
4
b
Alloc NH
Alloc NH
(CH2)3
NH2
NH2
(
Fmoc Arg
Pbf
CH )m
(CH )m
2
(CH2)3
2
a,e
f
N
CH2
C
O
Arg Nva Tyr Dap Hol
Pbf tBu Boc
N
CH CO
Fmoc Arg
Pbf
N
CH2
C
O
Arg Nva Tyr Dap Hol
N
CH2 CO
2
Pbf tBu Boc
Fmoc-YM3-m
O
O
C
NH
CH )m
NH
NH
(CH )m
NH
(
Fmoc Arg
Pbf
(CH2)3
Arg Nva Tyr Dap Hol CH2 CO
Pbf tBu Boc
(CH2)3
2
2
g
a,h
N
CH2
C
O
N
H
Arg
N
CH2
C
Arg Nva Tyr Dap Hol N CH2 CO NH2
O
c(YM3-m)
m=2,3,4,6
=
Fmoc-Rink-Amide MBHA Resin
Scheme 2. Synthesis of the c(YM3-m) compounds. Nomenclature, in the general name, c(YM3-m), m stands for the number of methylenes in the alkyl
chain of the AGBU at position 2 and c represents a cyclic peptide while the absence of c indicates a pre-cyclic peptide. Reagents and conditions: a,
20% piperidine; b, Fmoc-AA-OH, HBTU, HOBt, DIPEA; c, Ac2O,DIPEA; d, Fmoc-Hol-OH, HATU, HOAt, DIPEA; e, Fmoc-Arg(Pbf)-OH, HATU, HOAt, DIPEA; f,
(
PPh3)4Pd(0), N-methylmorpholine, AcOH; g, BTC, DIPEA; h, TFA, Triisopropylsilane, TDW.
Generally, coupling toasecondaryaminebystandardHBTUme-
analyzed. We observed a major difference between PFPC and the
diated procedures results in incomplete attachment. Previously,
effective coupling of Fmoc amino acids to secondary amines was
achieved by the use of bis(trichloromethyl) carbonate, (triphos-
gene, BTC) to form the Fmoc amino acid chlorides [45]. To ensure
reaction completion, elevated temperatures and long reaction
times were also used [9,25,45].
two phosgene derivatives. PFPC converted the pre-cyclic peptide
to urea cyclic peptide almost completely before the addition of
base, whilephosgeneandBTCledonlytopartialconversionbefore
theadditionof thebase(Figure 1(A–C)). Thisobservationreaffirms
the assumption that the first step in the mechanism of phosgene
and BTC mediated urea cyclization involves the formation of
N-alkylated omega ammonium chloride salt. A second portion
of peptidyl resin was sampled 1 h after adding the base. HPLC
indicated that the base addition contributed to the complete
conversion to the urea cyclic form only in the BTC and phosgene
mediated procedures (Figure 1(D–F)). There was a decrease in
product purity in the PFPC mediated urea cyclization compared
to BTC or phosgene mediated procedure. We decided to use
the BTC urea cyclization procedure as BTC is easier to handle
than phosgene. For c(YM3-m), more than 80% conversion to the
urea form was obtained for all peptides (Table 1). Urea cyclic
peptides were cleaved after Fmoc removal and purified using
HPLC. Analytical HPLC and high resolution mass spectrometry
were used to characterize the isolated compounds (Figure 2).
For the synthesis of the YM analogs, standard Fmoc protocols
on solid support [44] were used as shown in Scheme 2. All
couplingstepswereexecutedusingHBTUasthestandardcoupling
reagent, except coupling of Fmoc amino acids to the secondary
amine of AGBU, which was performed using HATU. HATU is more
reactivethanHBTUandallowscouplinginhighlyhinderedpeptide
sequences [46]. We managed to reduce the excess of reagents and
HATU to 1.5 equivalents without reducing the coupling yields. To
ensure complete conversion, longer coupling periods were used.
After the attachment of Fmoc-Arg(Pbf), the resin was divided
into four portions, and different AGBU (3a–d) were introduced to
form four peptides. Simultaneous removal of the orthogonal Alloc
protecting groups from both AGBU yielded the desired pre-cyclic
peptidyl resin with two N-alkylated omega amines. Optimization
of the cyclization step forming urea bridge was studied on a model
peptide and was implemented for all four peptides.
PKB/Akt Model
All backbone cyclic peptides were screened for inhibition of
PKB/Akt in a cell-free radioactive assay, and compared with
PTR6154 (Table 1). c(YM3-4) had poor anti PKB/Akt activity while
the other peptides preserved anti PKB/Akt potency. c(YM3-2)
and c(YM3-3) inhibited PKB/Akt activity in the same manner as
the parent peptide (0.92 and 0.59 µM, respectively). c(YM3-6)
significantlyimprovedtheinhibitoryeffectcomparedtotheparent
peptide (0.16 µM). The concept of conformational screening was
demonstratedinthisstudyoffourpeptides,whichdifferbygradual
increase of ring size.
Backbone Urea Cyclization
α
The N Fmoc-protected pre-cyclic peptide, Fmoc-YM3-3, was
selected as a model for optimization of the urea cyclization
conditions. The efficiency of several carbonyl donating reagents,
namelyphosgene,BTCanddipentafluorophenylcarbonate(PFPC),
for urea cyclization was compared. In addition, the effect of the
consecutive base addition approach previously described [32] on
urea backbone cyclization was evaluated. The reaction progress
was monitored using HPLC, and product formation was confirmed
using mass spectrometry. Three batches of 50 mg resin, loaded
with Fmoc-YM3-3, were swelled in dichloromethane and were
introduced with each of the above reagents. Samples from all
three batches were collected prior to the addition of the base.
Small portions of peptidyl resin were cleaved and samples were
Methods
The following abbreviations were used throughout the text. The
abbreviationsforaminoacidsareaccordingtotheIUPAC-IUBCom-
mission of Biochemical Nomenclature, http://www.chem.qmul.
www.interscience.com/journal/psc Copyright ꢀc 2010 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2010; 16: 178–185

NOVEL METHOD FOR THE SYNTHESIS OF UREA BACKBONE CYCLIC PEPTIDES
Figure 1. HPLC monitoring of urea cyclization optimization for YM3-3: (a) BTC 2 h (b) Phosgene 2 h (c) PFPC 2 h (d) BTC + DIPEA 1 h (e) Phosgene +
DIPEA 1 h (f) PFPC + DIPEA 1 h. Peak A represents the pre-cyclic peptide. Peak B represents urea backbone cyclic peptide.
+
+
+
2+
3+
Figure 2. Characterization of c(YM3-6): (a) analytical HPLC; (b) MALDI-TOF MS: MH , MNa , MK observed; (c) HRMS: MH2 , MH3 observed.
ac.uk/iupac/AminoAcid/. ACN, acetonitrile; Alloc, allyloxycarbonyl;
Boc, t-butoxycarbonyl; BTC, bis(trichloromethyl)carbonate; DI-
PEA, diisopropylethylamine; DMF, N,N-dimethylformamide; ESI,
electrospray ionization; EtOAc, ethyl acetate; Fmoc, 9-fluorenyl
methyloxycarbonyl; Fmoc-OSu, 9-fluorenylmethyloxycarbonyl-
N-hydroxysuccinimide; HATU, [2-(7-Aza-1H-benzotriazole-1-
yl)-1,1,3,3-tetramethyluronium hexafluorophosphate]; HBTU,
or Bruker 500 MHz spectrometer. Chemical shifts are reported
downfield, relative to internal solvent peaks. Coupling constants
J are reported in Hertz. HRMS spectra were recorded on
nanospray ionization LTQ orbitrap. MALDI-TOF spectra were
recorded on a PerSeptive Biosystems MALDI-TOF MS, using
α-cyano-4-hydroxycinnamic acid as matrix. TLC plates used were
aluminum sheets silica gel 60 F254. Column chromatography was
performed on silica gel 60 (230–400 mesh).
[
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluo-
rophosphate]; HOAt, 1-hydroxy-7-aza-benzotriazole; HOBt,1-
hydroxybenzotriazole; HRMS, high resolution mass spectrome-
try; MALDI, matrix assisted laser desorption ionization; MBHA,
methylbenzhydrylamine; NMR, nuclear magnetic resonance;
Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; PFPC,
pentafluorophenyl carbonate; RP-HPLC, reverse phase high pres-
sure liquid chromatography; SPPS, solid phase peptide synthesis;
tBu, tert-butyl; TDW, tri-distilled water; TFA, trifluoroacetic acid;
TLC, thin layer chromatography; TOF, time of flight.
AllanalyticalHPLCwererecordedat220 nmataflowof1 ml/min
on (A) RP-18 column (5 µ 75 × 4.6 mm), eluents A (0.1% TFA in
TDW) and B (0.1% TFA in ACN) were used in a linear gradient
(95% A → 5% A in 12 min) or (B) RP-18 column (5 µ 250 × 4.6 mm,
110 Å), eluents A (0.05% TFA in TDW) and B (ACN) were used in a
linear gradient (95% A → 5% A in 35 min). Preparative HPLC were
recorded at 220 nm on RP-18 column (10 µ 250 × 10 mm, 110 Å),
eluents A (0.05% TFA in TDW) and B (ACN) were used in a linear
gradientof(C) (85%A→65%Ain25 min)ataflowrateof5 ml/min.
Chemistry – General
General Procedure for the Preparation of Carbamates 1a–d
All starting materials were purchased from commercial sources
A solution of 1 mol alkylene diamine dissolved in 500 ml
chloroform was stirred and cooled to 0 C. A solution of 0.1 mol
allylchloroformate in 250 ml chloroform was added dropwise
1
◦
and were used without further purification. Routine H NMR
1
3
and C NMR spectra were acquired on Bruker 300, Bruker 400
J. Pept. Sci. 2010; 16: 178–185 Copyright ꢀc 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

HUREVICH ET AL.
Table 1. Characterization and IC50 of the c(YM3-m) analogs
Peptide
name
Ring
size
Calculated mass
Observed mass
[MH2 ]/2
Percentage urea
conversion (HPLC)
Percentage purity
(HPLC)
IC50 (µM)
(95% confidence)
[MH22
+
]
2+
m
PTR6154
c(YM3-2)
c(YM3-3)
c(YM3-4)
c(YM3-6)
0.94
(
(
(
0.78–1.14)
2
3
4
6
27
28
29
31
1046.6450
1060.6607
1074.6763
1102.7076
523.3220
530.3303
537.3373
551.3488
79
86
>95
>95
>95
>95
0.92
0.58–1.45)
0.59
0.48–0.73)
4.0
>95
90
(
2.8–5.7)
0.16
(0.03–0.77)
Observed mass was determined by HRMS. Purity was determined by analytical HPLC method B. Urea conversion was determined by preparative
HPLC method C. IC50 of PKB inhibition was determined according to radioactive kinase assay as described [48]. IC50 values and 95% confidence range
(
parenthesis) were determined using Graphpad Prism 5.
over 3 h and the reaction mixture was stirred overnight at room
temperature. For 1a–c, the organic layer was washed with water
until no trace of starting material was apparent in the TLC
General Procedure for the Preparation of N-Fmoc-[N-(Alloc)
ω-alkyl]glycine 3a–d
A stirred solution of 0.1 mol of 1a–d and 0.085 mol NaCNBH3
(32 : 8:1 CH2Cl2 –MeOH–AcOH), dried over Na2SO4 and solvent
◦
dissolvedin200 mlMeOHwascooledto0 C. Ameasuredquantity
was evaporated to yield colorless oil. For 1d, the crude was
extracted with aqueous HCl 1 M solution followed by the addition
of NaOH 4 M until pH >8. The solution was extracted with EtOAc,
dried over Na2SO4 and evaporated to yield a clear oil of 1d. The
resulting crude 1a–d proved pure enough for further use. When
higher purity is needed, the products can be purified using flash
chromatography (20% MeOH in CH2Cl2).
of 0.085 mol of glyoxylic acid monohydrate (0.85 equiv) was
added in portions and the reaction was stirred overnight at room
temperature. Solvent was evaporated to yield an oily residue of
2
a–d. Crude 2a–d was dissolved in 260 ml water followed by the
addition of 0.17 mol of Et3N and set to stir at room temperature
until the solution became clear. A solution of 0.075 mol Fmoc-OSu
(0.75 equiv in relation to 1a–d) in 400 ml ACN was added to
the reaction mixture. The reaction was stirred for 4 h while the
pH was kept alkaline by adding Et3N if necessary. The reaction
mixture was washed with petroleum ether (3 × 250 ml) and ether:
petroleum ether 7 : 3 (3 × 250 ml). The aqueous layer was cooled
and acidified to pH 3–4 with HCl 2 M, and extracted with EtOAc
(4×250 ml).TheorganiclayerwaswashedwithHCl1M (2×150 ml),
saturated KHSO4 (2 × 150 ml) and dried over Na2SO4. Solvent was
evaporated to yield colorless oil that later solidified upon cooling
to give a sticky white paste. Products 3a–c were recrystallized
from refluxing toluene to yield a white paste, which was collected
and washed extensively with cold petroleum ether to give a white
powder. Product 3d was recrystallized from a mixture of toluene
Allyl 2-aminoethylcarbamate 1a
Yield: 51%. 1H NMR (300 MHz, CDCl3): δ 1.60–1.75 (br, 2H),
2
.59–2.94 (t, J = 5.6, 2H), 2.99–3.38 (q, J = 5.8, 2H), 4.36–4.66
(
(
d, J = 5.1, 2H), 5.02–5.36 (m, 2H), 5.35–5.68 (br, 1H) 5.73–6.03
1
3
m, 1H). C NMR (101 MHz, CDCl3): δ 41.4, 43.2, 65.4, 117.5, 132.8,
56.5.
1
Allyl 3-aminopropylcarbamate 2a
Yield: 58%. 1H NMR (300 MHz, CDCl3): δ 1.55–1.63 (m, 2H),
1
4
5
6
.64–1.69 (br, 2H), 2.68–2.79 (t, J = 6.6, 2H), 3.16–3.29 (m, 2H),
.44–4.58 (d, J = 5.1, 2H), 5.10–5.29 (m, 2H), 5.36–5.58 (br, 1H),
and n-hexane. The mixture was left to crystallize overnight at
1
3
.79–5.94 (m, 1H). C NMR (101 MHz, CDCl3): δ 32.7, 38.9, 39.5,
◦
−
20 C and the crystals were collected and washed extensively
5.2, 117.3, 132.9, 156.3.
with cold petroleum ether to give white powder.
Allyl 4-aminobutylcarbamate 1c
Yield: 80%. 1H NMR (300 MHz, CDCl3) δ 1.40–1.80 (br, 6H),
N-Fmoc-[N-(Alloc)-aminoethyl]glycine 3a
2
.55–2.72 (t, J = 6.6, 2H), 3.0–3.2 (q, J = 5.6, 2H), 4.48–4.53
1
Yield: 80%. H NMR (400 MHz, CDCl3): δ 2.9–3.1 (br, 1H), 3.15–3.25
(
d, J = 5.4, 2H), 5.1–5.3 (m, 2H), 5.7–5.8 (br, 1H), 5.8–6.0 (m, 1H).
1
3
(br, 1H), 3.25–3.35 (br, 1H), 3.25–3.60 (br, 1H), 3.8–3.9 (s, 1H),
.9–4.0 (s, 1H), 4.1–4.3 (m, 1H), 4.4–4.7 (br, 4H), 5.0–5.3 (m, 2H),
.7–5.9 (m, 1H), 7.27–7.46 (m, 2H), 7.5–7.6 (m, 2H), 7.65–7.80 (m,
H). C NMR (101 MHz, DMSO-d6): δ 46.49 (46.55), 47.2 (47.7),
8.7 (49.0), 64.2 (64.25), 67.2, 116.78 (116.85), 120.1, 125.1, 127.10
C NMR (101 MHz, CDCl3): δ 27.2, 30.4, 40.7, 41.5, 65.2, 117.3,
32.9, 156.2.
3
5
2
4
1
1
3
Allyl 6-aminohexylcarbamate 1d
Yield: 75%. 1H NMR (300 MHz, CDCl3) δ 1.18–1.36 (m, 4H),
1
3
(127.15), 127.6, 133.6, 140.5 (140.7), 143.6 (143.7), 155.4 (155.6),
155.9, 171.0 (171.2). ES–MS mass calculated for C23H24N2O6
.37–1.53 (m, 4H), 1.70–1.97 (br, 2H), 2.54–2.78 (t, J = 7.0, 2H),
+
.03–3.22 (m, 2H), 4.39–4.62 (m, 2H,), 4.65–4.85 (br, 1H), 4.9–5.1
425.16 (MH ). Found 425.27. HRMS (NSI-orbitrap) calculated for
1
3
+
(br, 1H), 5.1–5.34 (m, 2H), 5.78–6.00 (m, 1H). C NMR (101 MHz,
C23H24N2NaO6 447.1532 (MNa ). Found 447.1528. HPLC purity
CDCl3): δ 26.45, 26.50, 29.9, 33.3, 40.9, 42.0, 65.6, 117.8, 133.3, 156.5.
>99% room temperature 12.58.
www.interscience.com/journal/psc Copyright ꢀc 2010 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2010; 16: 178–185

NOVEL METHOD FOR THE SYNTHESIS OF UREA BACKBONE CYCLIC PEPTIDES
N-Fmoc-(N-(Alloc)3-aminopropyl)glycine 3b
with CH2Cl2 (2 × 2 min) and dried in vacuum. A solution of 5%
AcOH and 2.5% N-methyl morpholine in CH2Cl2 was added under
Ar stream and Tetrakis (triphenylphosphine) palladium (0) (0.7
equiv) was added. The reaction was left to stir in the dark for
Yield: 74%. 1H NMR (400 MHz, CDCl3): δ 1.35–1.45 (br, 1H),
1
.55–1.75 (br, 1H), 2.85–2.95 (br, 1H), 2.95–3.11 (br, 1H), 3.11–3.25
(
(
(
br, 1H), 3.25–3.45 (br, 1H), 3.8–3.9 (s, 1H), 3.9–4.0 (s, 1H), 4.0–4.2
2
h and then washed with 0.5% DIPEA in NMP (3 × 5 min), 0.5%
m, 1H), 4.3–4.8(br, 4H), 5.1–5.4(br, 2H), 5.8–6.1(m, 1H), 7.27–7.45
sodium diethyldithiocarbamate trihydrate in NMP (5 × 2 min),
NMP (2 × 2 min) and CH2Cl2 (2 × 2 min). Cleavage: The resin
was washed with CH2Cl2 (2 × 2 min) and dried under vacuum. A
solution of 2.5% TDW and 2.5% triisopropylsilane in trifluoroacetic
acid (TFA) was added and the reaction proceeded for 3 h at room
temperature. The solution was separated by filtration and the
resin was rinsed with neat TFA. The TFA mixture was treated
with a cooled solution of ether : hexane 1 : 1 and peptides were
precipitated by centrifugation. Crude peptides were dissolved in
acetonitrile : TDW 1 : 1 solution and lyophilized.
1
3
br, 4H), 7.45–7.6 (m, 2H), 7.7–7.8 (m, 2H). C NMR (101 MHz,
DMSO-d6): δ 27.8 (28.1), 37.7 (37.8), 45.6 (45.9), 46.57 (46.61), 48.4
48.8), 64.1, 66.6, 67.0, 115.8, 119.97(120.02), 124.79(125.03), 127.1,
33.7, 140.6 (140.7), 143.6 (143.8), 155.34 (155.48), 155.8, 170.9
(
1
(
171.2). HRMS (NSI-orbitrap) calculated for C24H26N2O6 439.1791
+
(MH ). Found 439.1868. HPLC purity >99% room temperature
1
2.70.
N-Fmoc-[N-(Alloc)4-aminobutyl]glycine 3c
1
Urea cyclization BTC-mediated:
A
solution of BTC,
Yield: 60%. H NMR (400 MHz, CDCl3): δ 1.1–1.3 (br, 2H), 1.4–1.6
bis(trichloromethyl) carbonate (0.33 equiv) in CH2Cl2 was added
to the resin and left to stir. After 2 h, DIPEA (2 equiv) was added
and the reaction was left to stir overnight at room temperature.
The resin was washed with CH2Cl2 (2 × 2 min). Urea cyclization
PFPC mediated: A solution of PFPC (1 equiv) in CH2Cl2 was added
to the resin and left to stir. After 2 h, DIPEA (2 equiv) was added
and the reaction was left to stir overnight at room temperature.
The resin was washed with CH2Cl2 (2 × 2 min). Urea cyclization
phosgene mediated: A solution of 20% phosgene in toluene (1
equiv phosgene relative to resin) was added to resin preswelled
in CH2Cl2 and left to stir. After 2 h, DIPEA (2 equiv) was added and
the reaction was left to stir overnight at room temperature. The
resin was washed with CH Cl (2 × 2 min).
(
br, 1H), 2.9–3.5 (br, 4H), 3.8–3.9 (s, 1H), 3.9–4.0 (s, 1H), 4.15–4.25
(m, 1H), 4.35–4.65 (br, 4H), 5.1–5.4 (m, 4H), 5.85–6.1 (m, 1H),
1
3
7
.27–7.45 (m, 4H), 7.45–7.65 (m, 2H), 7.65–7.8 (m, 2H). C NMR
(101 MHz, DMSO-d6): δ 25.1 (25.5), 27.11 (27.23), 47.15 (47.26),
4
1
7.92 (48.22), 48.68 (49.08), 64.59, 66.97 (61.4), 117.28, 120.55,
25.20 (125.60), 127.50 (127.53), 128.01 (128.13), 134.34, 141.14
(141.36), 144.27 (144.46), 155.98, 156.39, 1s71.42 (171.70). HRMS
+
(
4
NSI-orbitrap) calculated for C25H28N2O6 453.1947 (MH ). Found
53.2025. HPLC purity >95% room temperature 12.88.
N-Fmoc-[N-(Alloc)6-aminohexyl]glycine 3d
1
Yield: 58%. H NMR (400 MHz, CDCl3): δ 1.0–1.5 (br, 8H), 3.0–3.2
2
2
c(YM3-2) was prepared from 150 mg of Fmoc-Rink Amide
MBHA resin. HPLC purity >95% room temperature (B) 16.48.
(
(
m, 3H), 3.2–3.35 (m, 1H), 3.8–3.9 (s, 1H), 3.9–4.0 (s, 1H), 4.1–4.3
m, 1H), 4.4–4.5 (m, 1H), 4.5–4.65 (br, 3H), 5.1–5.35 (m, 2H),
+
+
MS (MALDI-TOF): calculated for C46H81N18O10 1045.6378 (MH ),
found 1045.32. HRMS exact mass (ESI-orbitrap): calculated for
5
2
.8–6.0 (m, 1H), 7.27–7.45 (m, 4H), 7.5–7.65 (m, 2H), 7.65–7.80 (m,
1
3
H). C NMR (125 MHz, CDCl3): δ 26.18 (26.26), 27.5, 28.0, 29.55
2
+
2+
1046.6450, found (MH2 ) 523.3220; calculated
C46H82N18O10
(
(
29.71), 40.7 (40.8), 47.1, 48.3 (48.5), 48.9, 65.4, 67.3 (67.6), 117.40
117.57), 119.82, 124.69 (124.84), 126.9, 127.54 (127.57), 132.82,
3
+
3+
for C46H83N18O10 1047.6523, found (MH3 ) 349.2176.
c(YM3-3) was prepared from 150 mg of Fmoc-Rink Amide
MBHA resin. HPLC purity >95% room temperature (B) 16.39.
1
41.12 (141.26), 143.8, 155.97, 156.3 (156.5), 173.5 (173.6). HRMS
+
(NSI-orbitrap) calculated for C27H32N2O6 503.2158 (MNa ). Found
+
+
MS (MALDI-TOF): calculated for C47H83N18O10 1059.6534 (MH ),
found 1058.99. HRMS exact mass (ESI-orbitrap): calculated for
5
03.2153. HPLC purity >95% room temperature 13.50.
2
+
2+
1060.6607, found (MH2 ) 530.3303; calculated
C47H84N18O10
General Methods for SPPS
3
+
3+
for C47H85N18O10 1061.6680, found (MH3 ) 353.8897.
c(YM3-4) was prepared from 150 mg of Fmoc-Rink Amide
MBHA resin. HPLC purity >95% room temperature (B) 16.29.
Swelling: The resin was swelled for at least 2 h in CH2Cl2.
Fmoc removal: The resin was treated with a solution of 20%
piperidine in 1-methyl-2-pyrrolidinone (NMP) (2 × 20 min), and
thenwashedwithNMP(5×2 min).HBTUcoupling:Fmocprotected
amino acids (1.5 equiv) were dissolved in NMP. N,N-DIPEA (1.5
equiv) and 1-hydroxybenzotriazole (HOBt) were added and the
mixture was cooled to 0 C. [2-(1H-Benzotriazole-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate] (HBTU) (1.5 equiv)
was added and the mixture was preactivated by mixing for
+
+
MS (MALDI-TOF): calculated for C48H85N18O10 1073.6691 (MH ),
found 1073.75. HRMS exact mass (ESI-orbitrap): calculated for
2
+
2+
1074.6763, found (MH2 ) 537.3373; calculated
C48H86N18O10
3
+
3+
for C48H87N18O10 1075.6836, found (MH3 ) 358.5612.
c(YM3-6) was prepared from 150 mg of Fmoc-Rink Amide
MBHA resin. HPLC purity >95% room temperature (B) 17.67.
◦
+
+
MS (MALDI-TOF): calculated for C50H89N18O10 1101.7004 (MH ),
found 1101.60. HRMS exact mass (ESI-orbitrap): calculated for
1
0 min, added to the resin, and shaken for 1 h. The resin was
2
+ 2+
1102.7076, found (MH2 ) 551.3488; calculated
washed with NMP (3 × 2 min). Capping: The resin was treated
with a solution of Ac2O (10 equiv) and DIPEA (7.15 equiv) in
dimethylformamide (DMF) for 20 min and washed with NMP
C50H90N18O10
3
+
3+
forC50H91N18O10 1103.7149,found(MH3 )367.9018(Figure 2).
(
3×2 min).HATUcoupling:Fmocprotectedaminoacids(1.5equiv)
PKB Assay
were dissolved in NMP, DIPEA (1.5 equiv) and 1-hydroxy-7-aza-
benzotriazole (HOAt) were added and the mixture was cooled to
PKB kinase (HisꢀPHPKBEEEFlag) was prepared as described by
Klein et al. [47] except that for routine screening the enzyme was
only partially purified in one step on Ni-NTA agarose (Qiagen)
as described [47]. The radioactive kinase assay was as described
by Reuveni et al. [48] except that the reaction mix comprised
50 mM Hepes pH 7.4, 0.1 mM EGTA, 0.1% (v/v) 2-mercaptoethanol,
◦
0
C. [2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate] (HATU) (1.5 equiv) was added and the
mixture was preactivated by mixing for 10 min, added to the
resin, and shaken overnight at room temperature. The resin was
washed with NMP (3×2 min). Alloc removal: The resin was washed
J. Pept. Sci. 2010; 16: 178–185 Copyright ꢀc 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

HUREVICH ET AL.
1
0 mM magnesium acetate, 3-µM RPRTSSF peptide, 10 µM γ 32P-
8 Gazal S,Gelerman G,Ziv O,Karpov O,Litman P,Bracha M,Afargan M,
Gilon C. J. Med. Chem. 2002; 45: 1665–1671.
ATP (1 µCi/assay well), the inhibitory compound and 0.005 units
HisꢀPHPKBEEEFlag. Astocksolutionofeachpeptidewasprepared
andtheconcentrationwasdeterminedbyaUVspectrophotometer
as described [49]. For initial screening, compounds were tested at
three to four concentrations between 20 and 0.8 µM. Compounds
that showed significant inhibition at 1 µM or less were retested
and IC50 values and 95% confidence range were determined using
Graphpad Prism 5 (Table 1). PTR6154 was included in every assay,
as a standard.
9
Qvit N, Hatzubai A, Shalev DE, Friedler A, Ben-Neriah Y, Gilon C.
Biopolymers 2009; 91: 157–168.
1
0 Hess S, Linde Y, Ovadia O, Safrai E, Shalev DE, Swed A, Halbfinger E,
Lapidot T, Winkler I, Gabinet Y, Faier A, Yarden D, Xiang Z, Portillo FP,
Haskell-Luevano C, Gilon C, Hoffman A. J. Med. Chem. 2008; 51:
1026–1034.
1
1 Hess S, Ovadia O, Shalev DE, Senderovich H, Qadri B, Yehezkel T,
Salitra Y, Sheynis T, Jelinek R, Gilon C, Hoffman A. J.Med.Chem. 2007;
5
0: 6201–6211.
12 Byk G, Halle D, Zeltser I, Bitan G, Selinger Z, Gilon C. J. Med. Chem.
996; 39: 3174–3178.
3 Ghalit N, Poot AJ, Furstner A, Rijkers DTS, Liskamp RMJ. Org. Lett.
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1
1
1
2
Conclusions
4 Giblin MF, Wang N, Hoffman TJ, Jurisson SS, Quinn TP. Proc. Natl.
Acad. Sci. U. S. A. 1998; 95: 12814–12818.
There is a demand for chemical procedures that enable more
extensive screening of backbone cyclic peptides. We established
and optimized a new BTC mediated urea cyclization procedure
that can be used for the formation of urea backbone cyclic
peptides. This procedure allowed efficient connection of two
relatively distant amines through a backbone cyclic urea bridge
on solid support. The method was validated by synthesizing four
compounds of PKB/Akt substrate mimetics. Urea cyclization was
used to produce a backbone cyclic peptide that inhibited PKB/Akt
in the nano-molar range in vitro and improved the activity of
the parent peptide by almost tenfold. In addition, an improved
and efficient procedure for the preparation of a set of crystalline
15 Ruan FQ, Chen YQ, Itoh K, Sasaki T, Hopkins PB. J. Org. Chem. 1991;
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Strachan RG, Nutt RF, Arison BH, Homnick C, Randall WC, Glitzer MS,
Saperstein R, Hirschmann R. Nature 1981; 292: 55–58.
5
1
1
9
1
9 Cheng S, Craig WS, Mullen D, Tschopp JF, Dixon D, Pier-
schbacher MD. J. Med. Chem. 1994; 37: 1–8.
2
0 Kopple KD, Baures PW, Bean JW, Dambrosio CA, Hughes JL,
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α
orthogonally protected N (ω-functionalized alkylated) glycines
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into the peptide using standard Fmoc SPPS protocols. Three
reagents, phosgene, PFPC and BTC were assessed as the urea
bridge formation agent. BTC, which is relatively easy to store and
handle, was found to be an efficient reagent.
The methodology described here is general and is based on
a small set of building blocks. This approach can be used to
extend the diversity of backbone cyclic peptides and to increase
the probability of finding potent backbone cyclic peptides against
various targets.
23 Ali FE, Bennett DB, Calvo RR, Elliott JD, Hwang SM, Ku TW, Lago MA,
Nichols AJ, Romoff TT, Shah DH, Vasko JA, Wong AS, Yellin TO,
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2
2
2
2
4 Weckbecker G, Lewis I, Albert R, Schmid HA, Hoyer D, Bruns C. Nat.
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5 Barda Y, Cohen N, Lev V, Ben-Aroya N, Koch Y, Mishani E, Fridkin M,
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28 Filip K, Oleszczuk M, Pawlak D, Wojcik J, Chung NN, Schiller PW,
The synthetic improvements presented here will facilitate the
use of backbone cyclization by nonspecialist scientists, so that this
methodology may become a powerful tool in peptide-based drug
discovery research.
Izdebski J. J. Pept. Sci. 2003; 9: 649–657.
2
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3
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Acknowledgements
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A.L. was supported by grants from the Prostate Cancer Foundation
(PCF, USA) and The Goldhirsh Foundation (USA).
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J. Pept. Sci. 2010; 16: 178–185 Copyright ꢀc 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc