Synthesis of N′-substituted Ddz-protected hydrazines and their application in solid phase synthesis of aza-peptides

Article abstract of DOI:10.1016/j.tet.2008.11.038

Hydrazine derivatives are of considerable scientific and industrial value. Substituted hydrazines are precursors for many compounds of great interest and importance, among them aza-peptides. (Aza-peptides are peptide analogues in which one or more of the

Full text of DOI:10.1016/j.tet.2008.11.038

Tetrahedron 65 (2009) 1737–1745
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of N0-substituted Ddz-protected hydrazines and their application
in solid phase synthesis of aza-peptides
*
Noam S. Freeman, Mattan Hurevich, Chaim Gilon
Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 June 2008
Received in revised form 5 November 2008
Accepted 13 November 2008
Available online 18 November 2008
Hydrazine derivatives are of considerable scientific and industrial value. Substituted hydrazines are pre-
cursors for many compounds of great interest and importance, among them aza-peptides. (Aza-peptides
are peptide analogues in which one or more of the a-carbons, bearing the side chain residues, has been
replaced by a nitrogen atom.) Aza-amino acid residues conserve the pharmacophores necessary for bi-
ological activity while inducing conformational changes and increased resistance to proteolytic degra-
dation. These properties make aza-peptides attractive tools for structure–activity relationship studies and
drug design. We describe the synthesis of N0-substituted 2-(3,5-dimethoxyphenyl)propan-2-yloxy-
carbonyl (Ddz) protected hydrazines. A general approach for solid phase synthesis of aza-peptides has
been developed based on the in-situ activation of the N-Ddz,N0-substituted hydrazines with phosgene,
followed by introduction to the N-terminus of a resin-bound peptide. The Ddz-aza-amino building units
include aliphatic, aromatic and functionalized side chains, protected for synthesis by the Fmoc strategy.
Solid phase aza-peptide synthesis is demonstrated including selective mild deprotection of Ddz with
Mg(ClO4)2 and coupling of the next amino acid with triphosgene. Ddz deprotection is orthogonal with the
Fmoc and Boc protecting groups, making the solid phase Ddz-aza-peptide synthesis compatible with both
the Fmoc and the Boc strategies. The Ddz-protected hydrazines have wide applications in the synthesis of
substituted hydrazines and in the synthesis of aza containing peptidomimetics.
Keywords:
Substituted hydrazines
Ddz protecting group
Solid phase synthesis
Peptidomimetics
Aza-peptides
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
drugs, insecticides, reagents in organic chemistry of diverse func-
tions, precursors for synthesis of heterocycles,4 and in peptidomi-
Substituted hydrazines have found many scientific and com-
mercial applications.1,2 The importance of hydrazine derivatives is
reflected in the great number of such compounds synthesized to
date and the synthesis of substituted hydrazines has been the
subject of many studies.3 Hydrazine derivatives are employed as
metic synthesis.5 The vast interest in peptidomimetic structures is
enthused by their value to understand structure–activity relation-
ships and the ongoing search for in vivo active drug lead com-
pounds.6,7 Peptidomimetics are designed in an attempt to enhance
biological activity while overcoming undesirable peptide proper-
ties such as rapid metabolism by proteolysis, poor bioavailability
and nonselective receptor binding.8–10 Substituted hydrazines are
key components in the synthesis of aza-peptides,11–13 a peptidomi-
Abbreviations: ACN, acetonitrile; AcOH, acetic acid; Alloc, allyloxycarbonyl; Boc,
tert-butoxycarbonyl; BTC, bis(trichloromethyl)carbonate; Cbz, benzyloxycarbonyl;
DBE, 1,2-dibromoethane; CH2Cl2, dichloromethane; Ddz, 2-(3,5-dimethoxy-
phenyl)propan-2-yloxycarbonyl; DIEA, N,N-diisopropylethylamine; DMAP, 4-(N,N-
dimethylamino)pyridine; DMF, dimethylformamide; DMSO, dimethyl sulfoxide;
ES-MS, electron spray mass spectrometry; EtOAc, ethyl acetate; Fmoc, 9-fluo-
renylmethoxycarbonyl; HBTU, O-benzotriazol-1-yl-N,N,N0,N0-tetramethyluronium
hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; HPLC, high performance liq-
uid chromatography; HRMS, high resolution mass spectrometry; MBHA, methyl-
benzhydrylamine; MS, mass spectrometry; NMP, N-methyl-2-pyrrolidinone; NMR,
nuclear magnetic resonance; PE, petroleum ether (40–60); PyBOP, benzotriazole-1-
yl-oxy-trispyrrolidino-phosphonium hexafluorophosphate; RP, reverse phase; rt,
room temperature; SPPS, solid phase peptide synthesis; t-Bu, tert-butyl; TDW, triple
distilled water; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TIS, triisopropylsilane;
TLC, thin layer chromatography; Ts, para-toluenesulfonyl; UV, ultraviolet.
* Corresponding author. Tel.: þ972 2 6585276; fax: þ972 2 6416358.
metic in which one or more of the
a-carbons, bearing the side
chain residues, has been replaced by a nitrogen atom (Fig. 1).
Aza-amino acid residues impart special conformational proper-
ties14 to the parent peptide structure due to the loss of ster-
eogenicity and reduction of flexibility.
C-terminus
N-terminus
O
O
R
N
H
R
H
N
H
N
N
N
H
H
O
O
peptide
aza-peptide
R : side chain
Figure 1. Peptide and aza-peptide.
E-mail address: gilon@vms.huji.ac.il (C. Gilon).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.11.038

1738
N.S. Freeman et al. / Tetrahedron 65 (2009) 1737–1745
The incorporated aza-amino acid is evidently able to adopt the
O
2a-l
R1
proper pharmacophore orientation required for activity and se-
lectivity yet confers resistance towards proteolytic degradation.
Replacement of a Ca by N in a peptide results in an additional
possibility for the formation of hydrogen bonds and was also found
to affect the acidity of the neighbouring amide N–H bonds.13 The
considerable reduction of the flexibility, brought about by the in-
clusion of hydrazide and urea structural elements, has been shown
H
N
2. Reduction
NaCNBH3
1.
R1 R2
R2
Ddz
Ddz
Ddz
N
H
Aldehyde
or
3a-l
O
Ketone
NH2
MeO
O
N
H
1
H
N
R
X
2m
R
OMe
to induce
b
-turn conformations in aza-peptides.15,16 Hence, the
N
H
Alkyl halide
replacement of a certain Ca by N in a biologically active peptide will
most probably affect its conformation and hence its absorption,
transport, distribution, enzyme or receptor binding and metabolic
stability. The presence of an aza-amino acid residue may increase
the biological activity and/or improve the pharmacokinetic prop-
erties of the parent peptide. These properties suggest that aza-
peptides could be useful tools for peptidomimetic drug design and
for structure–activity relationship studies, and indeed, application
of aza-peptides in the study of biologically active peptides has had
significant success.11,17 In addition, an aza-amino acid scan of bi-
ologically active peptides was recently introduced as a method for
identifying biologically active conformations.16
3m
Scheme 1. Synthetic scheme of N0-alkyl,N-Ddz hydrazines 3a–m.
hydrazone, which was reduced without further purification. Re-
duction was performed with sodium cyanoborohydride under mild
acidic conditions achieved by acetic acid (methods B and C) or by
gradual addition of p-toluenesulfonic acid monohydrate solution
managed by means of indicator toning (method A).20,21 The
obtained BH2CN adduct was subsequently hydrolized with aq
NaOH in methanol to give the desired N0-substituted Ddz hydrazine
3a–l.
Synthetic difficulties were encountered in the reduction of the
aryl hydrazone derivatives. Reported reduction of Fmoc, Boc and
Cbz aryl hydrazones suggests that catalytic hydrogenation is pre-
ferred over reduction with sodium cyanoborohydride.15,21 How-
ever, other reports indicate that catalytic conditions and the
duration of the reaction substantially affect the yield and that care
must be taken to avoid over reduction.20,22 In the case of benzal-
dehyde hydrazone, 30 min of catalytic hydrogenation over 10%
palladium–carbon gave the desired aza-Phe precursor 3j in 74%
yield.y However, the same conditions did not suffice for reduction of
the 4-tert-butoxy benzaldehyde hydrazone to obtain the aza-Tyr-
(t-Bu) precursor 3k. Attempts to prepare 3j and 3k by method A
(THF, 1.5 mol equiv NaBH3CN, 1.1 mol equiv TsOH$H2O, indicator
toning) or by method B (1.5 mol equiv NaBH3CN, 2 mol equiv AcOH)
resulted in considerably long reaction times even with further ad-
dition of reducing agent. Ultimately, reaction of the benzaldehyde
hydrazone with excess reducing agent and excess acetic acid,
method C (3 mol equiv NaBH3CN, 5 mol equiv AcOH), gave com-
plete reduction overnight. These conditions were also successful in
preparation of the aryl derivatives 3k–l without apparent depro-
tection of the Ddz protecting group.
The 2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl (Ddz)
group is an attractive protecting group, which can be removed under
extremely mild conditions.18 The Ddz protecting group is normally
removed with dilute TFA solutions (0.2–3%) in a few minutes.
However, Ddz can also be selectively removed by photolysis or by
Lewis acids such as Mg(ClO4)2 or ZnCl2. Ddz deprotection during
solid phase peptide synthesis (SPPS) utilizing Mg(ClO4)2 in acetoni-
trile (ACN) at 50 ꢀC was found to be fully orthogonal with side chain
Boc protection. These extremely mild conditions offer an additional
degree of orthogonality and enable Ddz to replace Fmoc in the SPPS
Fmoc/t-Bu chemistry strategy.19 To our surprise, ourliterature search
came up with few substituted Ddz-protected hydrazines. For these
reasons we have chosen to prepare Ddz-protected substituted hy-
drazines and examine their application for the SPPS of aza-peptides.
In this report we present two general methods for the synthesis
of N0-substituted 2-(3,5-dimethoxyphenyl)propan-2-yl carbazates
(N0-substituted Ddz protected hydrazines). We then evaluate the
application of N-substituted Ddz hydrazines in solid phase aza-
peptide synthesis using acid labile Rink amide methylbenzhydryl-
amine (MBHA) resin with mild Lewis acid mediated Ddz
deprotection.
2. Results and discussion
2.3. Nucleophilic substitution with alkyl halide
2.1. Synthesis of N0-substituted 2-(3,5-dimethoxy-
phenyl)propan-2-yl carbazates
Reaction of Ddz hydrazide 1 with the appropriate alkyl halide,
tert-butyl bromoacetate and potassium carbonate in dry DMF gave
the desired Ddz substituted hydrazine 3m. Although dialkylation
was expected to be a major by-product, only mono alkylation was
observed. This method is still under investigation in our lab with
other alkyl halides. Recent reports demonstrated that nucleophilic
substitution of tert-butyl bromoacetate with Fmoc carbazate, per-
formed under different conditions, proceeded in considerably
lower yield (20%) than the corresponding Boc or Cbz carbazates
(80%).23 The above conditions gave the Aza-Asp(t-Bu) precursor in
62% yield.
Thirteen N0-substituted Ddz hydrazines were prepared in
satisfying yields (62–95%) using two synthetic methods: reductive
alkylation and nucleophilic addition. The conveniently orthogonal
removal of the Ddz protecting group provides these compounds
with a wide variety of applications in the synthesis of substituted
hydrazines.
Our synthesis of N0-substituted Ddz hydrazines was designed
according to the literature procedures for the synthesis of Boc,
Fmoc and Cbz substituted hydrazines.3,15,20–22Two general syn-
thetic pathways were used to prepare N0-substituted Ddz hydra-
zines 3a–m (Scheme 1). (1) Reduction of the Ddz hydrazones,
derived from the reaction of commercially available 2-(3,5-dime-
thoxyphenyl)propan-2-yl carbazate (Ddz hydrazide) 1 with either
aldehyde or ketone, to yield the desired N0-substituted Ddz
hydrazines 3a–l in satisfying yields (methods A–C, Table 1). (2)
Introduction of side chain corresponding to aspartic acid was
achieved by nucleophilic substitution of alkyl halide, tert-butyl
bromoacetate, with Ddz hydrazide to obtain compound 3m
(method D).
2.2. Reduction of Ddz hydrazones
y
Condensation of Ddz hydrazide 1 with the appropriate aldehyde
or ketone 2a–l in THF readily gave the corresponding Ddz
Amino acids abbreviations are according to the IUPAC-IUB Commission of
Biochemical Nomenclature (http://www.chem.qmul.ac.uk/iupac/AminoAcid).

N.S. Freeman et al. / Tetrahedron 65 (2009) 1737–1745
1739
Table 1
Summary of the synthesis of 3a–m
Entry
Structure of 2a–m
Structure of 3a–m
Method
A
Yield %
86
Precursor for the
incorporation of
H
N
O
H
a
Aza-norvaline
Aza-valine
Ddz
N
H
H
N
O
b
c
A
A
78
83
Ddz
N
H
O
H
H
Aza-homoisoleucine
(racemic)
N
Ddz
N
H
O
H
H
N
d
e
f
B
B
B
B
81
86
87
73
Aza-leucine
Ddz
Ddz
N
H
H
N
O
Aza-isoleucine (racemic)
Aza-homoleucine
Aza-ornithine(Alloc)
N
H
H
O
H
N
Ddz
N
H
O
H
N
Alloc
Boc
Alloc
Boc
g
Ddz
N
N
H
H
N
H
H
O
H
N
h
i
B
B
70
76
Aza-ornithine(Boc)
Aza-lysine(Boc)
Ddz
Ddz
N
N
H
N
H
H
H
Boc
N
H
H
N
N
OH
N
Boc
H
O
H
N
j
C
C
95
78
Aza-phenylalanine
H
Ddz
N
H
O
H
N
k
Aza-tyrosine(Ot-Bu)
H
Ddz
N
t-Bu
O
H
t-Bu
O
N
O
H
N
l
C
68
62
Aza-tryptophan(Boc)
H
Ddz
N
H
N
Boc
Boc
H
N
O
O
Br
t-Bu
m
D
Aza-aspartic acid(t-Bu)
Ddz
N
H
t-Bu
O
O
Method A: THF, aldehyde or ketone 1 mol equiv, NaBH3CN 1.5 mol equiv, TsOH$H2O 1.1 mol equiv, indicator toning. Method B: THF, aldehyde or ketone 1 mol equiv, NaBH3CN
1.5 mol equiv, AcOH 2 mol equiv. Method C: THF, aldehyde or ketone 1 mol equiv, NaBH3CN 3 mol equiv, AcOH 5 mol equiv. Method D: DMF, alkyl halide 1 mol equiv, K2CO3
1.1 mol equiv.
2.4. In-situ activation of N0-substituted Ddz hydrazines
and solid phase synthesis of aza-peptides (Fig. 2)
N0-substituted-N0-chlorocarbonyl Ddz hydrazines, which were
subsequently reacted with the free N-terminal amine of the pep-
tidyl-resin as activated aza-amino building units. After indication by
TLC that the activation reaction was complete (10 min), excess
phosgene and toluene were removed under reduced pressure and
the resulted activated aza-amino acid was introduced to the solid
supported deprotected peptide without further purification. This
coupling of activated aza-amino acid was performed with excess of
activated unit and DIEA in CH2Cl2 as previously described.15 All
couplings were monitored by Kaiser-ninhydrin26 and Chloranil27
tests and were repeated when tests showed positive free amine. The
Ddz protecting group was removed conveniently and selectively
with Mg(ClO4)2 in ACNat 50 ꢀC as previously described.19 Prior Fmoc
We examined the use of N0-substituted Ddz hydrazines in the
solid phase synthesis of aza-peptides as previously described for the
N0-alkyl Fmoc carbazates.15,24,25 Procedures for activation of N0-
substituted Ddz hydrazines and their coupling as activated aza-
amino acids to the free amine of peptidyl-resin are similar to those
reported for N0-alkyl Fmoc carbazates with minor modifications. N0-
Substituted Ddz hydrazines with excess diisopropyl ethylamine
(DIEA) in dry dichloromethane (CH2Cl2) at 0 ꢀC were slowly treated
with excess phosgene solution in toluene. These conditions con-
verted the N0-substituted Ddz hydrazines to their corresponding

1740
N.S. Freeman et al. / Tetrahedron 65 (2009) 1737–1745
O
H2N Peptide1
Resin-bound peptide
O
H
N
H
N
Cl
Cl
Ddz
N
Cl
R(P)
O
Ddz
Ddz
N
H
DIEA, CH2Cl2
R(P)
O
H
N
Mg(ClO4)2
H2N
Peptide1
N
N
H
Peptide1
N
N
H
CH3CN, 50 °C
R(P)
R(P)
R'(P)
H
O
Fmoc-amino acid, BTC
2,4,6-collidine, CH2Cl2
Fmoc
N
N
H
N
N
H
Peptide1
O
R(P)
1. Deprotection: 20% piperidine/NMP
2. Peptide elongation: Fmoc-amino acid,
HOBT, HBTU, DIEA
: Rink amide MBHA resin
R(P), R'(P) : Protected side chains
3.Cleavage: TFA, TIS, H2O (92.5/5/2.5, v/v/v)
R'
O
H
Peptide2
N
N
H
N
R
N
H
Peptide1 NH2
O
Aza-peptide
Figure 2. General protocol for solid phase synthesis of aza-peptides using N0-substituted Ddz hydrazines.
loading test28,29 of resin-bound Fmoc-phenylalanine ensured that
these conditions do not cause any apparent cleavage of substrate
from the resin. Removal of Ddz from the aza-amino acid residue was
monitored by UV absorbance of the deprotection solution18 and was
found to be slower than described for the removal of Ddz from
N-terminus amino acid. After 3 h the deprotection solution was
monitored and renewed every half an hour to ensure complete
deprotection. Difficult coupling of the next Fmoc-amino acid to the
aza-amino acid was achieved with bis(trichloromethyl)carbonate
(BTC) and 2,4,6-colidine in CH2Cl230 and subsequently repeated. The
Kaiser-ninhydrin test could not be used to monitor this coupling.
The Chloranil test occasionally gave red coloured beads instead of
the usual green colour. Aza-glycine seemed to give the fastest and
strongest response to colour the beads red, other aza-residues
reacted very slowly and only gave a faint red colour after several
hours up to a day. Consequently, the coupling to the aza-amino acid
was monitored by cleavage with TFA of a very small bead sample
(‘small cleavage’) followed by MS analysis of the crude obtained. If
N-terminal aza-peptide mass was observed, additional coupling
under more forcing conditions of 60 ꢀC in 1,2-dibromoethane (DBE)
overnight was performed and the aza-peptide was subsequently
acylated using acetic anhydride solution in DMF.
As a model, the aza-peptide sequence Fmoc-Lys-azaVal-Ala-Ala-
Phe-NH2 4 was chosen. Two parallel aza-peptides were synthesized
(1) using Ddz and (2) using Fmoc-protected isopropyl substituted
hydrazine as aza-Val building unit precursors. Quantitative Fmoc
substitution of the peptidyl-resin28,29 prior to cleavage indicated
that in both cases the aza-peptide was obtained in an overall yield
of 50%. Crude HPLC of both aza-peptides were very similar (Sup-
plementary data). The crude peptide was purified by preparative
HPLC and characterized by mass spectrometry (Supplementary
data).
Selective Alloc deprotection from aza-Orn(Alloc) precursor 3g and
subsequent guanidilation can afford the Aza-arginine residue as
previously described.15 This convenient procedure for solid phase
synthesis of aza-peptides using Ddz orthogonal protection facili-
tates the generation of novel peptidomimetics such as branched
aza-peptides and cyclic aza-peptides. In addition, Ddz depro-
tection is orthogonal with the Fmoc and Boc protecting groups,
making the solid phase Ddz-aza-peptide synthesis compatible
with both the Fmoc and the Boc strategies. Substituted Ddz hy-
drazines have a wide range of applications in the synthesis aza-
peptide, aza-peptoide and other aza-peptidomimetics. Of note is
the possible incorporation of these compounds in the synthesis of
aza-b
3-amino acids, which utilizes Fmoc, Boc and Cbz substituted
hydrazines.23
3. Conclusion
We present the synthesis of novel N0-substituted Ddz hydra-
zines in which the N0-substituents are mainly derived from the
side chains of amino acids. We believe these compounds will find
a wide use in peptidomimetic synthesis and in the general pro-
duction of substituted hydrazines. We demonstrate the in-
corporation of these aza-amino acid precursors into peptides to
afford aza-peptides using standard Fmoc chemistry SPPS. Con-
sidering the convenience of the presented procedure for aza-
peptide synthesis and the diversity of Ddz protecting group, this
method should be of general use in the synthesis of aza-peptides
as well as other aza-peptidomimetic structures. We reason that
the various possibilities for Ddz removal and applications brought
about in this study will promote a wider use of this attractive
protecting group.
We demonstrate here the utilization of N0-substituted Ddz
hydrazines in the synthesis of aza-peptides. Similar to the Fmoc
aza-peptide synthesis strategy,15,24,25 acylation of N0-substituted
Ddz hydrazines with phosgene may afford activated Ddz-protected
aza-amino acids possessing aliphatic (Val 3a, Nvl 3c, Leu 3d, Hol
3f) aromatic (Phe 3j, Tyr 3k, Trp 3l), basic (Orn 3h, Lys 3i) and
acidic (Asp 3m) side chains protected for the Fmoc/t-Bu SPPS
strategy. Activated aza-amino acids applied to resin-bound pep-
tides followed by selective Ddz deprotection and subsequent
routine peptide elongation afforded the desired aza-peptides.
4. Experimental section
4.1. Hazards
Phosgene solution and triphosgene, bis(trichloromethyl)-
carbonate (BTC), are highly toxic and may cause death by inhalation.
These substances should be handled in a well-ventilated hood with
extreme caution.

N.S. Freeman et al. / Tetrahedron 65 (2009) 1737–1745
1741
4.2. Reagents
described.33 The crude yellowish oil product was used without
further purification. Yield: 75%. 1H NMR (CDCl3, 400 MHz):
2.73 (t,
d
Ddz hydrazide 1 and Fmoc-protected amino acids were pur-
chased from Nova Biochemicals (Lau¨felfingen, Switzerland). Unless
otherwise noted, aldehydes and ketones 2 and other starting ma-
terials and reagents were purchased from commercial suppliers
and used without further purification. The 20% solution of phos-
gene in toluene was purchased from Fluka (Seelze, Germany). Rink
amide MBHA resin (200–400 mesh) was purchased from CBL
(Patras, Greece; catalog no. BR-1305, lot no. 2536) and loading was
determined to be 0.6 mmol/g. Solvents for organic synthesis, tri-
fluoroacetic acid (TFA) and solvents for high performance liquid
chromatography (HPLC) were purchased from Bio-Lab (Jerusalem,
Israel).
J¼5.8 Hz, 2H), 3.47 (q, J¼6.0 Hz, 2H), 4.53 (d, J¼5.3 Hz, 2H), 5.16 (br
s, 1H), 5.19 (dd, J¼10.3, 0.7 Hz, 1H), 5.28 (dd, J¼17.2, 1.3 Hz, 1H),
5.81–5.99 (m, 1H), 9.80 (s, 1H).
4.4.2. 3-(tert-Butyloxycarbonylamino)propanal 2h
Reduction of the corresponding Boc protected b-alanine Wein-
reb amide, [2-(N-methoxy-N-methylcarbamoyl)ethyl] carbamic
acid tert-butyl ester, with LiAlH4 was performed as previously de-
scribed.34,35 The crude colourless oil product was used without
further purification. Yield: 55%. 1H NMR (CDCl3, 300 MHz):
d 1.42 (s,
9H), 2.70 (t, J¼5.9 Hz, 2H), 3.42 (q, J¼5.9 Hz, 2H), 4.88 (br s, 1H),
9.80 (s, 1H).
4.3. General methods
4.4.3. 1-tert-Butyloxycarbonyl-2-hydroxypyrrolidine 2i36,15
Overnight hydrolysis of Boc protected 4-amino butyraldehyde
diethyl acetal in AcOH/water 2:1 v/v solution gave a colourless oil
that was used without further purification. Yield: 85%. 1H NMR
(DMSO-d6, 400 MHz): d 1.40 (s, 9H),1.59–1.70 (m,1H), 1.71–1.83 (m,
2H), 1.87–2.00 (m, 1H), 3.07–3.17 (m, 1H), 3.25–3.30 (m, 1H), 5.20–
5.32 (m, 1H), 5.51 (br d, 1H).
Thin layer chromatography (TLC) was performed on Merck F245
60 silica gel plates (Darmstadt, Germany). Visualization was ach-
ieved with UVlight when applicable ordeveloped byiodine staining.
Flash chromatography was performed using silica gel 60 (230–400
mesh) (Merck Darmstadt, Germany). Nuclear magnetic resonance
(NMR) spectra were recorded on Bruker AMX-300 MHz or Bruker
DRX-400 MHz spectrometers. High resolution mass spectrometry
(HRMS) was recorded on a Thermo Scientific LTQ Orbitrap mass
spectrometer. Electron spray mass spectrometry (ES-MS) was
recorded on a Finnigan ThermoQuest LCQ-DUO ion trap mass
spectrometer. MALDI-TOF mass spectrometry was recorded on
4.4.4. 1-tert-Butoxycarbonylindole-3-carboxaldehyde 2l37
Boc protection of indole-3-carboxaldehyde gave yellow solid
that was used without further purification. Yield: 96%. 1H NMR
(CDCl3, 300 MHz):
1.4 Hz, 1H), 8.23 (s, 1H), 8.29 (dd, J¼7.0, 2.0 Hz, 1H), 10.10 (s, 1H).
d
1.71 (s, 9H), 7.34–7.46 (m, 2H), 8.15 (dd, J¼7.3,
a
PerSeptive Biosystems Voyager-DE PRO BioSpectrometry
workstation. Analytical RP-HPLC analysis was performed on a Phe-
nomenex Gemini RP-C18 column (5
4.5. General method A: synthesis of N0-alkyl 2-(3,5-
dimethoxyphenyl)propan-2-yl carbazates (3a–c)
m
, 250ꢁ4.6 mm) using a Merck-
Hitachi LaChrom D6000 interface system equipped with L-4250
UV–vis detector set at 220 nm, with an L-6200 intelligent pump and
AS-4000 autosampler. Eluants A (0.05% TFA in TDW) and B (0.05%
TFA in ACN) were used in a 35 min linear gradient (5% B to 95% B)
and a flow of 1 mL/min. Semi-preparative RP-HPLC was performed
A solution of 2-(3,5-dimethoxyphenyl)propan-2-yl carbazate 1
and the appropriate aldehyde or ketone 2a–c (1 mol equiv for
substances with high bp or 1.5 mol equiv for substances easily re-
moved by vacuum) in dry THF (0.65 M) was stirred overnight at rt
and concentrated in vacuo to obtain the corresponding hydrazone,
which was subsequently used without further purification. The
hydrazone was dissolved in dry THF (0.15 M) and treated with
NaBH3CN (1.5 mol equiv) with vigorous stirring. A few granules of
bromocresol green were introduced and the resulting blue solution
was treated dropwise with p-toluenesulfonic acid monohydrate
(1.1 mol equiv) in THF (2 mL). Every addition of the acid solution
was performed after indicator toning to maintain the reaction pH
between 3.5 and 5.16,20,21 After the yellowish colour persisted for
1 h, the solvent was removed under reduced pressure and the
residue was partitioned between EtOAc (50 mL) and brine (50 mL).
The aqueous phase was extracted with EtOAc (3ꢁ25 mL). The
combined organic layers were washed with saturated aq NaHCO3
(50 mL) and brine (50 mL), dried over MgSO4 and concentrated
under reduced pressure. The residue was dissolved in MeOH
(10 mL), treated with 1 M NaOH (1.2 mol equiv) and stirred for 1 h
at rt. The solvent was removed under reduced pressure and the
residue was dissolved in EtOAc (50 mL), washed with brine (50 mL),
dried over MgSO4 and concentrated under reduced pressure to
provide the desired compound.
on a Phenomenex Gemini RP-C18 column (5
m, 250ꢁ10.0 mm) using
a Merck-Hitachi LaChrom D6000 interface system equipped with
L-4250 UV–vis detector set at 220 nm and an L-6200 intelligent
pump. Eluants A (0.05% TFA in TDW) and B (0.05% TFA in ACN) were
used in a 50 min linear gradient (5% B to 95% B) and a flow of
5 mL/min. UV–vis spectrometry was recorded on a Shimadzu
UV-1650PC spectrometer. IR spectra were recorded on a Bruker
Vector 22 spectrometer. Melting points were recorded on a Fisher-
Johns melting point apparatus.
4.4. Chemistry
Commercially available aldehydes and ketones 2a–f,j were used
without further purification. Aldehydes 2g–i,l were prepared by
known procedures:
of the aza-Orn precursors were prepared by reduction of their
corresponding
-alanine Weinreb amides31 with lithium alumin-
b-amino aldehydes 2g and 2h for the synthesis
b
ium hydride,32 1-tert-butyloxycarbonyl-2-hydroxypyrrolidine 2i
for the synthesis of the aza-Lys(Boc) precursor was prepared by
hydrolysis of Boc protected 4-amino butyraldehyde diethyl acetal,
and Boc protection of indole-3-carboxaldehyde gave aldehyde 2l
for the synthesis of aza-Trp(Boc) precursor. Condensation of 1-tert-
butyloxycarbonyl-2-hydroxypyrrolidine 2i with Ddz hydrazide 1, to
form the corresponding hydrazone, was carried out in the presence
of p-toluenesulfonic acid.
4.5.1. N0-Propyl-2-(3,5-dimethoxyphenyl)propan-2-yl
carbazate (3a)
Product of the reaction of Ddz hydrazide 1 (0.51 g, 2.0 mmol)
and propionaldehyde 2a (0.105 mL, 2.05 mmol). Purification by
flash chromatography on silica using petroleum ether (PE)/EtOAc
6:4 as eluant gave a white solid. Yield 86% (0.51 g); mp¼56–58 ꢀC;
Rf¼0.22 (PE/EtOAc 7:3). IR (KBr) nmax 3300, 2958, 1716, 1686,
4.4.1. 3-(Allyloxycarbonylamino)propanal 2g
Reduction of the corresponding Alloc protected
b-alanine
Weinreb amide, [2-(N-methoxy-N-methylcarbamoyl)ethyl] carba-
mic acid allyl ester, with LiAlH4 was performed as recently
1601 cmꢂ1. 1H NMR (DMSO-d6, 400 MHz):
1.28–1.41 (m, 2H), 1.65 (s, 6H), 2.58 (br t, 2H), 3.73 (s, 6H), 4.31 (br s,
d
0.83 (t, J¼7.3 Hz, 3H),

1742
N.S. Freeman et al. / Tetrahedron 65 (2009) 1737–1745
1H), 6.37 (t, J¼2.2 Hz, 1H), 6.47 (d, J¼1.6 Hz, 2H), 8.46 (br s, 1H). 13
C
1H), 6.35 (t, J¼2.3 Hz, 1H), 6.50 (d, J¼2.5 Hz, 2H). 13C NMR (CDCl3,
NMR (DMSO-d6, 100 MHz):
d
11.5, 20.5, 28.9, 52.7, 55.0, 79.7, 97.8,
100 MHz): d 20.5, 26.8, 29.0, 55.2, 59.8, 81.5, 98.4, 102.9, 148.7,
102.6, 149.4, 155.6, 160.1. HRMS calcd for C15H25N2O4 297.18088
(MHþ), found 297.18060.
156.0, 160.6. HRMS calcd for C16H27N2O4 311.19653 (MHþ), found
311.19641.
4.5.2. N0-Isopropyl-2-(3,5-dimethoxyphenyl)propan-2-yl
carbazate (3b)
4.6.2. (R,S)-N0-sec-Butyl-2-(3,5-dimethoxyphenyl)propan-2-yl
carbazate (3e)
Product of the reaction of Ddz hydrazide 1 (0.51 g, 2.0 mmol)
and acetone 2b (0.151 mL, 2.05 mmol). Purification by flash chro-
matography on silica using PE/EtOAc 7:3 as eluant gave a white
solid. Yield 78% (0.46 g); mp¼88–89 ꢀC; Rf¼0.19 (PE/EtOAc 7:3). IR
(KBr) nmax 3283, 2979, 1725, 1600 cmꢂ1. 1H NMR (CDCl3, 400 MHz):
Product of the reaction of Ddz hydrazide 1 (0.51 g, 2.0 mmol)
and ethyl methyl ketone 2e (0.270 mL, 3.0 mmol). Product was
recrystallized from DMF/water to give a white solid. Yield 86%
(0.53 g); mp¼53 ꢀC. IR (KBr) nmax 3304, 3283, 2981, 2949, 1728,
1599 cmꢂ1. 1H NMR (CDCl3, 400 MHz):
d
0.87 (t, J¼7.4 Hz, 3H), 0.97
d
1.07 (d, J¼6.2 Hz, 6H), 1.75 (s, 6H), 3.16–3.26 (m, 1H), 3.78 (s, 6H),
(d, J¼6.0 Hz, 3H), 1.18–132 (m, 1H), 1.41–153 (m, 1H), 1.74 (s, 6H),
2.83–2.93 (m, 1H), 3.77 (s, 6H), 3.95 (br s, 1H), 6.35 (t, J¼2.1 Hz, 1H),
6.38 (br s, 1H), 6.50 (d, J¼2.3 Hz, 2H). 13C NMR (CDCl3, 100 MHz):
6.35 (t, J¼2.2 Hz, 1H), 6.50 (d, J¼2.1 Hz, 2H). 13C NMR (CDCl3,
100 MHz):
d 19.8, 28.9, 51.7, 55.3, 82.3, 98.6, 102.9, 148.4, 155.7,
160.7. HRMS calcd for C15H25N2O4 297.18088 (MHþ), found
297.18063.
d 9.9, 17.6, 27.3, 28.91, 28.99, 55.1, 56.4, 81.4, 98.4, 102.8, 148.7, 156.0,
160.6. HRMS calcd for C16H27N2O4 311.19653 (MHþ), found
311.19635.
4.5.3. (R,S)-N0-(2-Methyl-butyl)-2-(3,5-dimethoxyphenyl)propan-
2-yl carbazate (3c)
Product of the reaction of Ddz hydrazide 1 (0.25 g, 1.0 mmol)
and (R,S)-2-methyl butyraldehyde 2c (0.108 mL, 1.01 mmol). Puri-
fication by flash chromatography on silica using PE/EtOAc 8:2 as
eluant gave a white solid. Yield 83% (0.27 g); mp¼68 ꢀC; Rf¼0.50
(PE/EtOAc 7:3). IR (KBr) nmax 3277, 2961, 2873, 1721, 1599 cmꢂ1. 1H
4.6.3. N0-Isopentyl-2-(3,5-dimethoxyphenyl)propan-2-yl
carbazate (3f)
Product of the reaction of Ddz hydrazide 1 (0.76 g, 3.0 mmol)
and isovaleraldehyde 2f (0.483 mL, 4.5 mmol). Product was
recrystallized from DMF/water to give a white solid. Yield 87%
(0.85); mp¼66 ꢀC. IR (KBr) nmax 3316, 2954, 1690, 1600 cmꢂ1
.
1H
NMR (DMSO-d6, 400 MHz):
d
0.75–0.93 (m, 6H), 0.99–1.11 (m, 1H),
NMR (CDCl3, 400 MHz):
d
0.87 (d, J¼6.5 Hz, 6H), 1.27–1.35 (m, 2H),
1.32–1.47 (m, 2H), 1.65 (s, 6H), 2.35–2.44 (m, 1H), 2.51–2.59 (m, 1H),
1.55–166 (m, 1H), 1.74 (s, 6H), 2.81 (t, J¼7.0 Hz, 2H), 3.78 (s, 6H),
3.73 (s, 6H), 4.28 (br s, 1H), 6.37 (t, J¼2.2 Hz, 1H), 6.47 (d, J¼1.8 Hz,
6.29 (br s, 1H), 6.35 (t, J¼2.3 Hz, 1H), 6.50 (d, J¼2.3 Hz, 2H). 13C NMR
2H), 8.46 (br s, 1H). 13C NMR (DMSO-d6, 100 MHz):
d
11.0, 17.4, 26.7,
(CDCl3, 100 MHz): d 22.6, 25.9, 28.9, 36.7, 50.3, 55.2, 81.5, 98.3,
29.0, 32.5, 54.9, 56.9, 79.6, 97.7, 102.6, 149.3, 155.6, 160.1. HRMS
calcd for C17H29N2O4 325.21218 (MHþ), found 325.21198.
102.9, 148.7, 156.0, 160.6. HRMS calcd for C17H29N2O4 325.21218
(MHþ), found 325.21201.
4.6. General method B: synthesis of N0-alkyl 2-(3,5-
dimethoxyphenyl)propan-2-yl carbazates (3d–i)
4.6.4. N0-1-(3-(tert-Allyloxycarbonylamino)propyl)-2-(3,5-
dimethoxyphenyl)propan-2-yl carbazate (3g)
Product of the reaction of Ddz hydrazide 1 (0.64 g, 2.5 mmol) and
3-(tert-3-(allyloxycarbonylamino))propanal 2g (0.39 g, 2.5 mmol).
Purification by flash chromatography on silica using hexane/EtOAc
7:3 as eluant gave a yellowish oil. Yield 73% (0.72 g); Rf¼0.15 (hex-
ane/EtOAc 1:1). IR (KBr) nmax 3318, 2939, 1705, 1599 cmꢂ1. 1H NMR
A solution of 2-(3,5-dimethoxyphenyl)propan-2-yl carbazate 1
and the appropriate aldehyde or ketone 2d–i (1 mol equiv for
substances with high bp or 1.5 mol equiv for substances easily re-
moved by evaporation under reduced pressure) in dry THF (0.65 M)
was stirred overnight at rt and concentrated under reduced pres-
sure to obtain the corresponding hydrazone, which was sub-
sequently used without further purification. The hydrazone was
dissolved in dry THF (0.15 M) and treated with NaBH3CN
(1.5 mol equiv) with vigorous stirring. To this mixture, acetic acid
(2 mol equiv) was added and the reaction was stirred overnight at
rt. Additional NaBH3CN was added if necessary to ensure comple-
tion of the reaction verified by TLC (PE/EA 1:1). The solvent was
removed by evaporation under reduced pressure and the residue
was partitioned between EtOAc (50 mL) and brine (50 mL). The
organic layer was washed with saturated aq NaHCO3 (50 mL) and
brine (50 mL), dried over MgSO4 and concentrated under reduced
pressure. The residue was dissolved in MeOH (10 mL), treated with
1 M NaOH (1.2 mol equiv) and stirred for 1 h at rt. The solvent was
removed by reduced pressure and the residue was dissolved in
EtOAc (50 mL), washed with brine (50 mL), dried over MgSO4 and
concentrated under reduced pressure to provide the desired
compound.
(CDCl3, 400 MHz):
d 1.58–168 (m, 2H), 1.73 (s, 6H), 2.88 (br t,
J¼6.5 Hz, 2H), 3.16–3.28 (m, 2H), 3.77 (s, 6H), 4.53 (d, J¼5.2 Hz, 2H),
5.19 (dd, J¼10.4, 1.3 Hz, 1H), 5.26 (br s, 1H), 5.28 (dd, J¼17.2, 1.5 Hz,
1H), 5.84–5.96 (m, 1H), 6.34 (t, J¼2.2 Hz, 1H), 6.49 (d, J¼2.3 Hz, 2H).
13C NMR (CDCl3, 100 MHz):
d 27.2, 28.9, 38.9, 49.4, 55.2, 65.4, 81.9,
98.3, 102.9, 117.5, 132.9, 148.5, 155.8, 156.3, 160.6. HRMS calcd for
C19H30N3O6 396.21291 (MHþ), found 396.21289.
4.6.5. N0-1-(3-(tert-Butyloxycarbonylamino)propyl)-2-(3,5-
dimethoxyphenyl)propan-2-yl carbazate (3h)
Product of the reaction of Ddz hydrazide 1 (0.61 g, 2.4 mmol)
and 3-(tert-Butyloxycarbonylamino)propanal 2h (0.41 g 2.4 mmol).
Purification by flash chromatography on silica using 1% MeOH in
CH2Cl2 as eluant gave a white solid. Yield 70% (0.69 g); mp¼86–
89 ꢀC; Rf¼0.17 (hexane/EtOAc 1:1). IR (KBr) nmax 3318, 2981, 2949,
1692, 1600 cmꢂ1. 1H NMR (CDCl3, 400 MHz):
d 1.42 (s, 9H), 1.55–165
(m, 2H), 1.73 (s, 6H), 2.85 (t, J¼6.1 Hz, 2H), 3.15 (br s, 2H), 3.77 (s,
6H), 4.93 (br s, 1H), 6.34 (t, J¼2.3 Hz, 1H), 6.48 (d, J¼2.3 Hz, 2H). 13C
NMR (CDCl3, 100 MHz): d 27.6, 28.3, 28.9, 38.5, 49.4, 55.2, 79.0, 81.7,
4.6.1. N0-Isobutyl-2-(3,5-dimethoxyphenyl)propan-2-yl
carbazate (3d)
98.3, 102.9, 148.6, 155.9, 156.0, 160.6. HRMS calcd for C20H34N3O6
412.24421 (MHþ), found 412.24408.
Product of the reaction of Ddz hydrazide 1 (0.51 g, 2.0 mmol)
and isobutyraldehyde 2d (0.275 mL, 3 mmol). Product was recrys-
tallized from DMF/water to give a white solid. Yield 81% (0.50 g);
mp¼55 ꢀC. IR (KBr) nmax 3295, 2931, 2971, 1727, 1600 cmꢂ1. 1H NMR
4.6.6. N0-1-(4-(tert-Butyloxycarbonylamino)butyl)-2-(3,5-
dimethoxyphenyl)propan-2-yl carbazate (3i)
Product of the reaction of Ddz hydrazide 1 (0.45 g, 1.76 mmol)
and 1-tert-Butyloxycarbonyl-2-hydroxypyrrolidine 2i (0.33 g,
1.76 mmol). Formation of the hydrazone was catalyzed by
(CDCl3, 400 MHz):
d
0.90 (d, J¼6.9 Hz, 6H),1.66–1.78 (m,1H),1.75 (s,
6H), 2.63 (d, J¼5.8 Hz, 2H), 3.78 (s, 6H), 3.91 (br s, 1H), 6.19 (br s,

N.S. Freeman et al. / Tetrahedron 65 (2009) 1737–1745
1743
p-toluenesulfonic acid monohydrate (33 mg, 0.17 mmol). Purifica-
tion by flash chromatography on silica using hexane/EtOAc 7:3 as
eluant gave a yellowish oil. Yield 76% (0.57 g); Rf¼0.21 (hexane/
EtOAc 1:1). IR (KBr) nmax 3335, 2976, 2935,1699,1599 cmꢂ1. 1H NMR
hexane/EtOAc 8:2 as eluant gave a colourless oil. Yield 68% (0.66 g);
Rf¼0.65 (hexane/EtOAc 1:1). IR (KBr) nmax 3356, 2979, 2936, 1732,
1598 cmꢂ1. 1H NMR (CDCl3, 400 MHz):
d 1.66 (s, 9H), 1.72 (s, 6H),
3.75 (s, 6H), 4.22 (s, 2H), 6.33 (t, J¼2.2 Hz, 1H), 6.50 (d, J¼2.1 Hz,
2H), 7.22–7.27 (m, 1H), 7.29–7.34 (m, 1H), 7.64 (s, 1H), 7.70 (d,
J¼7.8 Hz, 1H), 8.13 (d, J¼7.8 Hz, 1H). 13C NMR (CDCl3, 100 MHz):
(CDCl3, 400 MHz):
d 1.36–1.52 (m, 13H), 1.72 (s, 6H), 2.79 (br t,
J¼6.0 Hz, 2H), 3.09 (br q, J¼6.0 Hz, 2H), 3.76 (s, 6H), 3.92 (br s, 1H),
4.71 (br s,1H), 6.33 (t, J¼2.2 Hz,1H), 6.45 (br s,1H), 6.48 (d, J¼2.2 Hz,
d
28.2, 28.8, 46.5, 55.2, 82.4, 83.8, 98.4, 102.9, 115.2, 119.4, 122.8,
2H). 13C NMR (CDCl3, 100 MHz):
d
24.8, 27.5, 28.3, 28.9, 40.3, 51.4,
124.6, 125.6, 129.8, 135.6, 148.3, 149.5, 155.6, 160.6, 160.7. HRMS
calcd for C26H34N3O6 484.24421 (MHþ), found 484.24350.
55.1, 78.9, 81.5, 98.2, 102.9, 148.6, 155.9, 156.0, 160.6. HRMS calcd for
C21H36N3O6 425.25986 (MHþ), found 425.25937.
4.8. General method D: synthesis of N0-alkyl 2-(3,5-
dimethoxyphenyl)propan-2-yl carbazates (3m)
4.7. General method C: synthesis of N0-aryl 2-(3,5-
dimethoxyphenyl)propan-2-yl carbazates (3j–l)
A well stirred suspension of 2-(3,5-dimethoxyphenyl)propan-
2-yl carbazate 1 and K2CO3 (1.1 mol equiv) in dry DMF (0.35 M) was
cooled down to 0 ꢀC in an ice bath. Then, alkyl halide (1 mol equiv)
was added, the ice bath was removed after 10 min and the reaction
mixture was stirred overnight at rt. The reaction mixture was di-
luted with water (50 mL) and extracted with EtOAc (3ꢁ30 mL). The
combined organic extracts were washed with water (50 mL) and
brine (50 mL), dried over MgSO4 and concentrated under reduced
pressure to provide the desired compound.
A solution of 2-(3,5-dimethoxyphenyl)propan-2-yl carbazate 1
and aldehyde or ketone 2j–l (1 mol equiv) in dry THF (0.65 M) was
stirred overnight at rt and concentrated in vacuo to obtain the cor-
responding hydrazone, which was subsequently used without fur-
ther purification. The hydrazone was dissolved in dry THF (0.15 M)
and treated with NaBH3CN (3 mol equiv) with vigorous stirring. To
this mixture, acetic acid (5 mol equiv) was added and the reaction
was stirred for 24 h. Additional NaBH3CN was added if necessary to
ensure completion of the reaction as verified by TLC (hexane/EA 1:1
or CH2Cl2/MeOH/TEA 98.5:1:0.5). The solvent was removed by
evaporation under reduced pressure and the residue was parti-
tioned between EtOAc (50 mL) and brine (50 mL). The organic layer
was washed with saturated aq NaHCO3 (50 mL) and brine (50 mL),
dried over MgSO4 and concentrated under reduced pressure. The
residue was dissolved in MeOH (10 mL), treated with 1 M NaOH
(1.2 mol equiv) and stirred for 1 h at rt. The solvent was removed by
reduced pressure and the residue was dissolved in EtOAc (50 mL),
washed with brine (50 mL), dried over MgSO4 and concentrated
under reduced pressure to provide the desired compound.
4.8.1. N0-(tert-Butyl acetate)-2-(3,5-dimethoxyphenyl)propan-2-yl
carbazate (3m)
Product of the reaction of Ddz hydrazide 1 (0.38 g, 1.5 mmol)
and tert-butyl bromoacetate 2m (0.22 mL, 1.5 mmol). Purification
by flash chromatography on silica using PE/EtOAc 7:3 as eluant gave
a white solid. Yield 62% (0.34 g); mp¼97–104 ꢀC; Rf¼0.46 (hexane/
EtOAc 1:1). IR (KBr) nmax 3368, 3316, 2978, 2939, 1742, 1709,
1595 cmꢂ1 1H NMR (CDCl3, 400 MHz):
. d 1.46 (s, 9H), 1.74 (s, 6H),
3.53 (s, 2H), 3.78 (s, 6H), 4.05 (br s, 1H), 6.34 (t, J¼2.3 Hz, 1H), 6.50
(d, J¼2.3 Hz, 2H), 6.73 (br s, 1H). 13C NMR (CDCl3, 100 MHz):
d 28.1,
28.8, 53.3, 55.2, 81.9, 98.4, 102.9, 148.5, 155.4, 160.6, 170.0. HRMS
4.7.1. N0-Benzyl-2-(3,5-dimethoxyphenyl)propan-2-yl
carbazate (3j)
calcd for C18H29N2O6 369.20201 (MHþ), found 369.20169.
Product of the reaction of Ddz hydrazide 1 (0.66 g, 2.6 mmol)
and benzaldehyde 2j (0.26 mL, 2.6 mmol). Purification by flash
chromatography on silica using 1% MeOH in CH2Cl2 as eluant gave
a colourless oil. Yield 95% (0.85 g); Rf¼0.58 (hexane/EtOAc 1:1). IR
4.9. General procedures for solid phase peptide synthesis
Solid phase reactions were performed in a manual glass reaction
vessel with an outer glass blanket to enable heating and cooling
and equipped with a sintered glass bottom and Teflon valve for
draining. All solid phase reactions were performed using Rink
amide MBHA resin and reactions were carried out in an amount of
solvent that was enough to cover the resin (0.1–0.15 M).
(KBr) nmax 3311, 2980, 2937, 1717, 1598 cmꢂ1 1H NMR (CDCl3,
.
400 MHz):
d
1.74 (s, 6H), 3.77 (s, 6H), 3.94 (s, 2H), 6.36 (t, J¼2.2 Hz,
1H), 6.51 (d, J¼2.2 Hz, 2H), 7.22–7.39 (m, 5H). 13C NMR (CDCl3,
100 MHz):
d 28.9, 55.2, 55.5, 81.8, 98.3, 102.9, 127.6, 128.4, 129.1,
137.0, 148.5, 155.8, 160.7. HRMS calcd for C19H25N2O4 345.18088
Solid phase peptide synthesis was carried out using the stan-
dard Fmoc/t-Bu strategy. The Fmoc protecting group was removed
by piperidine solution in NMP. Standard Fmoc-amino acid coupling
reactions were performed with HBTU/HOBt and DIEA in NMP/
CH2Cl2 mixture. Cleavage of aza-peptides and deprotection of side
chain protecting groups was carried out in TFA/TIS/H2O solution.
Cleavage of aza-peptides containing an aza-amino acid with an
aromatic side chain was performed at 0 ꢀC to avoid loss of aromatic
side chain promoted by acidic cleavage conditions.15 Crude
aza-peptides were precipitated with Et2O/hexane (1/1 mixture),
dissolved in ACN/H2O solution and lyophilized to obtain isolated
aza-peptides as white foams.
(MHþ), found 345.18079.
4.7.2. N0-(4-(tert-Butoxy)-benzyl)-2-(3,5-dimethoxyphenyl)-
propan-2-yl carbazate (3k)
Product of the reaction of Ddz hydrazide 1 (0.76 g, 3.0 mmol)
and 4-(tert-butoxy)-benzaldehyde 2k (0.522 mL, 3.0 mmol). Puri-
fication by flash chromatography on silica using 1% MeOH in CH2Cl2
as eluant gave a colourless oil. Yield 78% (0.97 g); Rf¼0.56 (hexane/
EtOAc 1:1). IR (KBr) nmax 3383, 3334, 2978, 2933, 1719, 1598 cmꢂ1
.
1H NMR (CDCl3, 400 MHz):
d 1.33 (s, 9H), 1.74 (s, 6H), 3.77 (s, 6H),
3.94 (s, 2H), 6.35 (t, J¼2.3 Hz, 1H), 6.50 (d, J¼2.1 Hz, 2H), 6.94 (d,
J¼8.4 Hz, 2H), 7.23 (d, J¼8.4 Hz, 2H). 13C NMR (CDCl3, 100 MHz):
d
28.8, 28.9, 55.1, 55.2, 78.5, 82.2, 98.4, 103.0, 124.1, 129.9, 130.7,
4.9.1. Fmoc deprotection
148.4, 155.2, 155.6, 160.7. HRMS calcd for C23H33N2O5 417.23840
(MHþ), found 417.23785.
The resin was treated twice with a 20% solution of piperidine in
N-methyl-2-pyrrolidinone (NMP) for 20 min each. The resin was
then washed with NMP (5ꢁ) to remove the Fmoc by-products
(dibenzofulvene and its piperidine adduct) and residual piperidine.
4.7.3. N0-3-((N-Boc-Indolyl)methyl)-2-(3,5-dimethoxyphenyl)-
propan-2-yl carbazate (3l)
Product of the reaction of Ddz hydrazide 1 (0.51 g, 2.0 mmol)
and 1-tert-butoxycarbonylindole-3-carboxaldehyde 2l (0.49 g,
2.0 mmol). Purification by flash chromatography on silica using
4.9.2. HOBt/HBTU coupling
Fmoc-protected amino acid (1.5 equiv), 1-hydroxybenzotriazole
hydrate (HOBt) (1.5 equiv) and diisopropyl-ethylamine (DIEA)

1744
N.S. Freeman et al. / Tetrahedron 65 (2009) 1737–1745
(1.5 equiv) were dissolved in NMP (about 7.5 vol) at rt. The solu-
tion was chilled to 0–5 ꢀC and O-benzotriazol-1-yl-N,N,N0,N0-tet-
ramethyluronium hexafluorophosphate (HBTU) (1.5 equiv) was
added followed by stirring for 10 min to dissolve. The solution of
activated amino acid was applied to the resin in CH2Cl2 (about 2.5
vol). The reaction was agitated for 1 h, the resin was then drained
and washed with NMP (3ꢁ). Coupling completion was monitored
by Kaiser-ninhydrin and Chloranil tests as described below. If
a positive ninhydrin or chloranil test was observed after 1 h, the
coupling reactionwas repeated using 1 equiv of activated amino acid.
(6 mol equiv relative to peptidyl-resin) was added and the solution
was applied to the vessel containing resin-bound N-terminal pep-
tide. The reaction mixture was shaken overnight, the resin was
drained and washed with CH2Cl2 (2ꢁ). The described Ddz-aza-
amino acid coupling was repeated once more. Coupling was mon-
itored by Kaiser-ninhydrin and Chloranil tests as described above.
4.11. General procedure for Ddz removal
The resin was washed with ACN (2ꢁ), Mg(ClO4)2 (10 mol equiv
relative to resin) was dissolved in ACN and applied to the vessel,
heated to 50 ꢀC and shaken for 1.5 h. The resin was washed with
ACN (2ꢁ), CH2Cl2 (2ꢁ), ACN (2ꢁ), and this procedure was repeated
once more. The procedure was further repeated for 0.5 h till no UV
absorption of the deprotection solution is visible on TLC plate. The
resin is then washed with ACN (3ꢁ) and CH2Cl2 (2ꢁ).
4.9.3. Capping
The resin was treated twice for 30 min each with a mixture of
acetic anhydride (0.5 M), DIEA (0.125 M) and HOBt (0.015 M) in
DMF. The resin was subsequently washed with DMF (3ꢁ) and
CH2Cl2 (3ꢁ).
4.9.4. Kaiser-ninhydrin test
4.12. General procedure for coupling of amino acid
to the N-terminus aza-amino acid peptidyl-resin
A sample containing 1–3 mg of the resin was withdrawn, placed
into a test tube and washed clean with ethanol, which was removed
by decantation. To the sample were added 3 drops of 80% phenol in
ethanol (w/v) solution, 4 drops of 1 mL of 1 mM aq KCN in 49 mL of
pyridine solution and 3 drops of 5% ninhydrin in ethanol (w/v)
solution. The solution was mixed well and placed in a preheated
heating block at 110 ꢀC for 5 min. A blue or violet colour of solution
is a positive indication for the presence of free amines, indicating
deprotection of the Fmoc protecting group or incomplete coupling
reaction.26
Difficult coupling to aza-amino acid was achieved according to
the procedure previously described using Fmoc-amino acid chlo-
rides:30 Fmoc-amino acid-OH (3 mol equiv relative to resin) and
bis-(trichloromethyl) carbonate (BTC, triphosgene) (1 mol equiv)
were suspended in CH2Cl2 and cooled down to 0 ꢀC in an ice bath.
2,4,6-Collidine (14 mol equiv) was added and after all the solids
were dissolved (about 1 min), the solution was poured onto the
resin and shaken for 3 h at rt. The resin was drained and this cou-
pling procedure was repeated once more. At the end of the second
coupling cycle, the resin was drained and washed with CH2Cl2 (5ꢁ).
The coupling was monitored by small cleavage followed by MS
analysis of the crude obtained. If N-terminus aza-peptide (starting
material) mass was observed, the above procedure was repeated
using 5 mol equiv Fmoc-amino acid in DBE at 60 ꢀC overnight and
the aza-peptide was subsequently capped.
4.9.5. Chloranil test
A sample containing 1–3 mg of the resin was withdrawn, placed
into a test tube and washed clean with ethanol, which was removed
by decantation. To the sample were added 3 drops of 2% acetal-
dehyde in DMF and 3 drops of 2% chloranil in DMF. The solution was
agitated at rt for 5 min. A green or blue colour of the beads is
a positive indication for the presence of free amines, indicating
deprotection of the Fmoc protecting group or incomplete coupling
reaction.27
4.12.1. Peptide cleavage from the resin and removal of side chain
protecting groups
Prior to cleavage, the peptidyl-resin was washed with CH2Cl2
(2ꢁ) and MeOH (2ꢁ) and thoroughly dried under reduced pressure.
4.9.6. Small cleavage
A sample of 5–10 mg peptidyl-resin was treated with a TFA/
TDW/TIS (95:2.5:2.5, v/v/v) solution precooled to 0 ꢀC. The solution
was shaken for 0.5 h at rt. The resin was removed by filtration and
the solvents were evaporated by a stream of nitrogen. The residue
was dissolved in ACN/TDW 1:1 solution and subsequently analyzed
by MS and HPLC.
A freshly made solution of TFA/TDW/triisopropylsilane (TIS)
(95:2.5:2.5, v/v/v) was cooled to 0 ꢀC (14 mL/g peptidyl-resin). The
resin was added, the mixture was maintained in the ice bath for
30 min and was subsequently shaken for 3 h at rt. The resin was
removed by filtration, washed with a small amount of neat TFA and
the combined TFA filtrates were concentrated (to about 2 mL) un-
der a stream of nitrogen. The oily residue was treated with a pre-
viously cooled to 0 ꢀC mixture of diethylether/hexane (1:1, v/v) and
the ether-hexane was decanted. The peptide precipitate was dis-
solved in ACN/TDW 1:1 and lyophilized. This complete cleavage
process as well as the small cleavage of aza-peptides containing an
aza-amino acid with aromatic side chain was carried out at 0 ꢀC as
previously recommended.15
4.9.7. Loading test
Quantitative Fmoc substitution of the resin was determined by
Fmoc cleavage and optical density measurements at 304 nm
according to Novabiochem’s proposed procedure.28,29
4.10. General procedure for introduction of an aza-amino
acid on solid support by the Ddz method
Acknowledgements
N0-Alkyl 2-(3,5-dimethoxyphenyl)propan-2-yl carbazate
3
(3 mol equiv relative to peptidyl-resin) was dissolved in dry CH2Cl2
(0.1 M) and DIEA (6 mol equiv) was added. The reaction mixture
was cooled to 0 ꢀC in an ice bath under argon and phosgene in
toluene (20% solution, 6 mol equiv) was added dropwise. Slow
addition of the phosgene solution, low temperature and basic
conditions during activation are necessary to avoid deprotection of
the acid labile Ddz protecting group. After completion (w10 min
observed by TLC (hexane/EA 1:1)), the excess phosgene and
solvents were evaporated under reduced pressure. The obtained
Ddz-aza-amino acid chloride was dissolved in dry CH2Cl2, DIEA
Authors thank Prof. Silvio E. Biali and Dr. Yaniv Barda for their
good advice.
Supplementary data
1H and 13C NMR spectral data for compounds 3a–m, analytical
HPLC and mass spectra for aza-peptide 4 are provided. This mate-
rial is available free of charges via the internet. Supplementary data
associated with this article can be found in the online version, at
doi:10.1016/j.tet.2008.11.038.

N.S. Freeman et al. / Tetrahedron 65 (2009) 1737–1745
17. Zega, A. Curr. Med. Chem. 2005, 12, 589–597.
1745
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