N-Nosyl-α-amino acids in solution phase peptide synthesis

Article abstract of DOI:10.1016/j.tet.2007.05.121

A highly efficient and practical synthesis of peptides in solution phase has been developed. The procedure is based on the use of p-nitrobenzenesulfonyl (nosyl) group for the protection of the amino function of α-amino acids. Every step of the procedure,

Full text of DOI:10.1016/j.tet.2007.05.121

Tetrahedron 63 (2007) 8164–8173
N-Nosyl-a-amino acids in solution phase peptide synthesis
*
Antonella Leggio, Maria Luisa Di Gioia, Francesca Perri and Angelo Liguori
Dipartimento di Scienze Farmaceutiche, Universitaꢀ della Calabria, Via Ponte P. Bucci cubo 15/C,
I-87036 Arcavacata di Rende (CS), Italy
Received 8 February 2007; revised 11 May 2007; accepted 31 May 2007
Available online 6 June 2007
Abstract—A highly efficient and practical synthesis of peptides in solution phase has been developed. The procedure is based on the use of p-
nitrobenzenesulfonyl (nosyl) group for the protection of the amino function of a-amino acids. Every step of the procedure, protection of the
amino function by the nosyl group, formation of the peptide bond, and removal of the sulfonamide group, is characterized by high yields and
excellent purity of the final products. The described strategy allows the preparation of short peptide sequences keeping the chiral integrity of
amino acid precursors. Compatibility of nosyl group with the side-chain protecting groups used in Fmoc-based strategy is demonstrated. The
method here presented is an alternative strategy that could provide advantages for future peptide synthesis.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
The use of p-nitrobenzenesulfonyl (nosyl) group to protect
the amino function is of fundamental importance for obtain-
ing N-methylated amino acids and peptides.6 Therefore, of
particular interest is the combination of the nosyl chemistry
with Fmoc chemistry when the aim of the synthetic work is
obtaining N-methylated peptides. In fact, the nosyl amino
acids introduced in a specific position of the peptide chain
are easily methylated on the a-amino function.6b
In peptide chemistry the choice of amino protecting group of
a-amino acids is critical. Many different protecting groups
have been developed to protect the amino function of a-ami-
no acids in peptide synthesis, but the use of carbamates has
received considerable attention due to their ability to mini-
mize base-catalyzed racemization during peptide synthesis.1
Moreover, strategies for peptide synthesis based on the use
of tert-butyl carbamate (Boc) and 9-fluorenylmethyl carba-
mate (Fmoc) are consolidated both in solution and solid
phases.
2. Results and discussion
The present work reports a new and alternative strategy for
carryingout peptide synthesisusingthenosylgroupto protect
the a-amino function and keeping on the side chain of amino
acid protecting groups compatible with the Fmoc group.
Existing methodology based on the Boc protecting group re-
quires trifluoroacetic acid for each step of Boc deprotection,
and an acidic environment with harsher chemicals to remove
side-chain protecting groups.2
The critical step when using the nosyl group concerns with
the removal of the sulfonyl group probably by nucleophilic
aromatic substitution (SNAr) with thiolate as nucleophile
as reported for N-alkylated derivatives.6c With N-nosyl-N-
methyl-a-amino acids, the deprotection of the amino func-
tion occurs easily at controlled temperature and in a short
time using the reagent system mercaptoacetic acid/sodium
methoxide.6a,b When the amino function is not methylated,
the removal of the nosyl group is not so easy. In fact, the hy-
drogen atom on the sulfonamide function is relatively acidic
and could reduce the efficiency of the sulfur nucleophile in
the nucleophilic aromatic substitution that provides the un-
masked amino function.
The Fmoc3,4 group is a base-labile protecting group that is
readily cleaved by a variety of amines via base-promoted
b-elimination; furthermore, acid-labile protecting groups
can be removed in its presence using milder acidic reagents.
However, Fmoc chemistry is better suited in solid phase than
in solution due to the problems connected with the deprotec-
tion process. In fact, dibenzofulvene, released during Fmoc
cleavage, can be inefficiently trapped by the base leading to
alkene polymerization and resultant problems in purifica-
tion.5
Keywords: N-Nosyl-a-amino acids; Solution phase peptide synthesis; N-
Nosyl-dipeptides; Mercaptoacetic acid.
The feasibility of every step of planned methodology, in-
cluding the protection of the a-amino acid, the elongation
* Corresponding author. Tel.: +39 0984492042; fax: +39 0984492855;
e-mail: a.liguori@unical.it
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.05.121

A. Leggio et al. / Tetrahedron 63 (2007) 8164–8173
Table 1. Results of the synthesis of N-nosyl-dipeptides 4a and 4b
8165
of the peptide chain, and deprotection of the terminal amino
function, was previously assessed using lipophilic amino
acid methyl esters of both D and L series as appropriate model
compounds.
Entry
R1
R2
R3
Yielda (%)
4a
4b
CH(CH3)2
H
H
CH(CH3)2
CH3
CH3
78
71
a
Isolated yield.
In a typical experiment, N-nosyl-L-valine (1a) and N-
nosyl-D-valine (1b) methyl esters were prepared by treating
the corresponding a-amino acid methyl ester hydrochlorides
with p-nitrobenzenesulfonyl chloride (Ns-Cl).6a Then, to
deprotect the amino function, 1a and 1b were treated with
3 equiv of mercaptoacetic acid in the presence of 8 equiv of
sodium methoxide in acetonitrile and methanol solu-
tion at 50 ꢀC.6a The unblocking reactions of 1a and 1b
proceeded at a markedly reduced rate. Typically, the reaction
time for all the deprotection processes of N-methylated N-no-
syl-a-amino acid methyl esters by the reagent system mer-
captoacetic acid/sodium methoxide was less than 10 min.6a
GC/MS analysis performed using the same mixture of 4a
and 4b showed the presence of two peaks corresponding to
the two diastereomers. The comparison of 1H NMR spectra
and GC/MS analyses of the mixture to those obtained from
the single crude products 4a and 4b excluded the formation
of epimerized products.
Diastereomeric tripeptides were also synthesized in order to
explore the possibility of obtaining longer peptide chains,
and to evaluate the stereochemical aspects of the entire
process that consists of removing the nosyl group from the
terminal amino function and coupling the deprotected pep-
tide with another N-nosyl-a-amino acid. N-Nosyl-dipeptides
4a and 4b were then deprotected on the terminal amino func-
tion using 3 equiv of mercaptoacetic acid and 16 equiv of
sodium methoxide (Scheme 2).
When the experiment was repeated using a larger amount of
sodium methoxide and under reflux the nosyl group was re-
moved more rapidly. In fact, treatment of N-nosyl-L-valine
methyl ester 1a with 3 equiv of mercaptoacetic acid in the
presence of 12 equiv of sodium methoxide (Scheme 1) pro-
vided the corresponding L-valine methyl ester 2a in 1 h. GC/
MS analysis performed on an aliquot of the crude reaction
product after acidic work up and treatment with diazo-
methane clearly showed the presence of the deprotection
coproduct, methyl 2-(4-nitrophenylthio)acetate.7 The for-
mation of this compound supports the hypothesis that the
removal of the nosyl group proceeds through a nucleophilic
aromatic substitution as reported for N-alkylated ana-
logues.6c Also N-nosyl-D-valine methyl ester 1b was depro-
tected in the same way (Scheme 1).
The deprotection reaction of the dipeptide systems required
an increased amount of sodium methoxide and went to com-
pletion in 2 h. After this time the deprotected dipeptides 5a
and 5b were treated with N-nosyl-L-isoleucine chloride6b
6
(Scheme 2). The tripeptides 7a and 7b were recovered in
89% and 84% yields, respectively (Table 2).
The diastereomeric tripeptides 7a and 7b were readily re-
solved by GC/MS (Fig. 2). Furthermore, the chromatograms
recorded with the crude products 7a and 7b compared to that
obtained from an appropriately prepared mixture of the two
diastereoisomers excluded any detectable racemization
process (Fig. 2).
R1
R2
O
R1
R2
O
OCH3
HSCH2COOH/ CH3ONa,
MeOH, CH3CN, reflux
OCH3
NsHN
H2N
2a-b
1a-b
1H NMR spectra of both crude products 7a and 7b showed
the presence of signals corresponding to only one diastereo-
mer. In particular the peaks of the amide and the methyl ester
protons of the C-terminal residue were selected to determine
the presence of the other diastereomer in each spectrum. The
corresponding signals were found with different chemical
shifts in two diastereomers as also showed by 1H NMR spec-
trum of a mixture of both 7a and 7b. Therefore, the obtained
compounds showed retention of configuration of the chiral
carbon atoms of the precursors.
R3
H
NaHCO3
CHCl3
Cl
NsHN
O
3
O
S
O
R1
O
R2
O
O2N
= Ns
NsHN
R3
OCH3
N
H
H
4a-b
Scheme 1.
Following these initial results, it seemed to be very attractive
to investigate the full applicability of the proposed method-
ology to prepare short peptide chains using, as starting
materials, N-nosyl-a-amino acids with functional groups in
their side chains.
The two deprotected products 2a and 2b were not isolated
and directly coupled with N-nosyl-D-alanine chloride6b
3
6b
in a chloroform solution containing aqueous NaHCO3 to
obtain the corresponding diastereomeric dipeptides Ns-D-
Ala-L-Val-OMe 4a and Ns-D-Ala-D-Val-OMe 4b (Scheme
1
1 and Table 1). The H NMR analysis of both single crude
In particular, we examined the possibility of obtaining
peptides containing a-amino acids protected on their side
chain with suitable protecting groups, which should resist
the conditions of protection and deprotection of the a-amino
function, and formation of the peptide bond. To this end,
acid-labile protecting groups were chosen to mask the func-
tional groups of the a-amino acid side chains.
products 4a and 4b revealed the presence of signals corre-
sponding to only one diastereomer. Furthermore, in the
1H NMR spectrum of an appropriately prepared mixture of
the two diastereomers 4a and 4b (Fig. 1), distinct signals
were observed for sulfonamidic and methyl ester protons
of the two diastereomers.

8166
A. Leggio et al. / Tetrahedron 63 (2007) 8164–8173
0
0.9
0.8
0.7
6.5
6.0
ppm
3.8
3.7
3.6
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0 ppm
Figure 1. 1H NMR spectrum of a mixture of Na-Ns-D-Ala-L-Val-OMe (4a) and Na-Ns-D-Ala-D-Val-OMe (4b).
Table 2. Results of the synthesis of N-nosyl tripeptides 7a and 7b
O
R3
O
HSCH2CO2H/
NaOCH3
MeOH, CH3CN,
reflux
O
R3
O
H
H
H
N
R1
4a-b
H
N
R1
Entry R1
R2
R3
R4
Yielda (%)
NsHN
OCH3
H2N
OCH3
R2
R2
7a
7b
CH(CH3)2
H
H CH3 CH(CH3)CH2CH3 89
CH(CH3)2 CH3 CH(CH3)CH2CH3 84
5a-b
R4
N
H
O
a
Isolated yield.
NaHCO3
CHCl3
Cl
Ns
H
6
The reaction was then extended to the amino acids 8b–d and
provided the corresponding N-nosyl-a-amino acids 9b–d in
75–89% overall yields (Table 3). The synthesis of nosyl-
dipeptides 11a–d was then accomplished by coupling of N-
nosyl-a-amino acids 9a–d with a-amino acid methyl ester
hydrochlorides 10a and 10b (Scheme 4) in the presence of
dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, and N-
methylmorpholine.Thecouplingreactionwasinitiallycarried
out by treating Na-nosyl-N3-Boc-L-lysine 9a dissolved in dry
tetrahydrofuran (THF) with L-alanine methyl ester hydrochlo-
ride 10a at 0 ꢀC (Scheme 4). After 2 h, the resulting dipeptide
Na-nosyl-N3-Boc-L-lysinyl-L-alanineOMe11awas isolated in
quantitative yield (99%) as the sole reaction product.
O
O
R3
H
O
S
O
H
N
R1
NsHN
R4
O2N
=
Ns
N
OCH3
R2
H
H
O
7a-b
Scheme 2.
The formation of the peptide bond was accomplished with
the aid of coupling reagents to prevent the partial deprotec-
tion of side-chain functional groups during the formation of
N-nosyl-a-amino acid chlorides by thionyl chloride.6b
Therefore, N-nosyl-a-amino acids 9a–d were prepared
using the corresponding a-amino acids 8a–d protected on
their side chains with acid-labile protecting groups
(Scheme 3).
1H NMR spectroscopy and mass spectra of the crude product
confirmed the structure of the dipeptide 11a, in particular,
1
the H NMR spectrum showed two signals at d 7.10 and
6.40 ppm corresponding to amidic and sulfonamidic pro-
tons, respectively.
In a typical experiment, N3-Boc-L-lysine 8a, chosen as
model compound, was dissolved in water/dioxane 1:1 and
treated with p-nitrobenzenesulfonyl chloride (Ns-Cl) in the
presence of triethylamine at 0 ꢀC (Scheme 3). The reaction
was completed within 30 min and afforded, after acidic
treatment with a 5% aqueous solution of KHSO4, Na-
nosyl-N3-Boc-L-lysine (9a) in 73% overall yield (Table 3).
The coupling reaction was also extended to the N-nosyl-a-
amino acids 9b–d (Scheme 4 and Table 4). The correspond-
ing dipeptides 11b–d were recovered in high yields (82–
1
95%) and purity as confirmed by H NMR and 13C NMR
1
spectra. H NMR spectra showed also the preservation of
the enantiomeric integrity of the chiral centers.

A. Leggio et al. / Tetrahedron 63 (2007) 8164–8173
8167
37.02
[A]
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
35.73
Time-->
26.00
26.00
28.00
28.00
30.00
30.00
32.00
32.00
34.00
34.00
36.00
38.00
38.00
40.00
40.00
42.00
42.00
44.00
44.00
[B]
0
36.00
Time-->
[C]
0
Time-->
26.00
28.00
30.00
32.00
34.00
36.00
38.00
40.00
42.00
44.00
Figure 2. Comparison of gas chromatograms: [A] GC/MS analysis of a mixture (30:70) of N-nosyl-L-Ile-D-Ala-L-Val-OCH3 (35.73 min) and N-nosyl-L-Ile-D-
Ala-D-Val-OCH3 (37.02 min); [B] GC/MS analysis of 7a; [C] GC/MS analysis of 7b.
R1
R1
H
O
S
O
H
OH
1) H2O/Dioxane/Et3N
2) KHSO4
OH
+
O2N
Cl
H2N
NsHN
9a-d
O
O
8a-d
O
O2N
S
= Ns
O
Scheme 3.
Therefore, the dipeptide Na-nosyl-N3-Boc-L-lysinyl-L-alani-
neOMe 11a dissolved in acetonitrile/methanol was depro-
tected at the terminal amino function by the reagent system
mercaptoacetic acid/sodium methoxide in the molar ratio
3:16 at reflux. The deprotection reaction was completewithin
1 h, after this time the deprotected product 12a was coupled,
with no purification beyond the acid extraction, with Na-no-
syl-L-valine chloride (13) in a chloroform solution containing
aqueous Na2CO3 (Scheme 5 and Table 5). After 30 min the
corresponding tripeptide Na-nosyl-L-valyl-N3-Boc-L-lysi-
nyl-L-alanineOMe 14a was recovered in 85% overall yield.
Table 3. Results of the synthesis of side-chain protected N-nosyl amino
acids 9a–d
Entry
R1
Yielda (%)
9a
9b
9c
9d
(CH2)4NH-(Boc)
CH2C6H4O-(t-Bu)
CH2S-(Trt)
73
75
83
89
CH2CONH-(Trt)
a
Isolated yield.
The selective deprotection of the terminal amino function of
dipeptides 11a–c to obtain subsequently the N-nosyl-tripep-
tides 14a–c was also investigated.
Analysis of the 1H NMR spectrum of crude product showed
the presence of signals referring to the protons of only one

8168
A. Leggio et al. / Tetrahedron 63 (2007) 8164–8173
R2
H
R2
H
R1
O
H
DCC/HOBt/
N-Me-Morpholine
THF
OCH3
NsHN
OCH3
OH
Cl
+
H3N
N
NsHN
H
R1
H
O
O
O
9a-d
11a-d
10a-b
a: R2 = CH3
b: R2 = CH(CH3)2
O
O2N
S
O
= Ns
Scheme 4.
Table 4. Results of the syntheses of side-chain protected Na-nosyl-dipeptide
methyl esters 11a–d
Table 5. Results of the syntheses of N-nosyl-tripeptide methyl esters 14a–c
Entry R1
R2
R3
Yielda (%)
Entry
R1
R2
Yielda (%)
14a
14b
14c
(CH2)4NH-(Boc) CH3
CH(CH3)2
CH2C6H4O-(t-Bu) CH(CH3)2 CH(CH3)CH2CH3 75
85
11a
11b
11c
11d
(CH2)4NH-(Boc)
CH2C6H4O-(t-Bu)
CH2S-(Trt)
CH3
99
82
87
95
CH(CH3)2
CH3
CH3
CH2S-(Trt)
CH3
CH(CH3)2
71
a
Isolated yield.
CH2CONH-(Trt)
a
Isolated yield.
R2
O
R2
O
H
H
O
HSCH2CO2H/ NaOCH3
NsHN
OCH3
H2N
OCH3
N
H
N
MeOH, CH3CN, reflux
R1
H
R1
H
H
O
11a-c
12a-c
R3
H
O
Cl
Na2CO3
CHCl3
Ns
13: R3 = (CH3)2CH
N
H
6: R3 = CH(CH3)CH2CH3
13, 6
O
S
O
R2
H
R3
NsHN
O
H
H
N
=
OCH3
O2N
Ns
N
H
R1
H
O
O
14a-c
Scheme 5.
diastereomer. Particularly diagnostic for the structure of 14a
are the signals corresponding to the amidic protons of ala-
nine and lysine at d 7.10 and 7.30 ppm, respectively, and
those ones corresponding to the sulfonamidic and urethanic
protons at d 6.36 and 4.92 ppm, respectively. Also the single
signal at d 3.75 ppm relative to the methyl ester protons con-
firmed the presence of a single diastereomer.
The highly reactive and easily prepared6b nosyl-protected
amino acid chlorides, used as reagents for peptide coupling
with lipophilic amino acids, allow a practical and simple
elongation of peptide chains.
The reagent system mercaptoacetic acid/sodium methoxide
used for the removal of the nosyl group permits the efficient
and specific deprotection of the a-amino function keeping
the protecting group on the side chains.
Afterward, tripeptides 14b and 14c were synthesized using
the same protocol adopted for the preparation of 14a
(Scheme 5 and Table 5). H NMR spectroscopic analysis
1
The method described is particularly attractive because the
adopted conditions, in every step of the entire process, do
not cause any loss of the optical integrity at the chiral centers
of the peptide.
of tripeptides 14a–c excluded the deprotection of side-chain
functional groups. In fact, no discernible extraneous signals
1
were observed in the H NMR spectra that revealed only
signals referring to the structure of the tripeptides 14a–c.
The successful application of the developed methodology to
the synthesis of peptides containing side-chain functional-
ized a-amino acids makes this method general for the solu-
tion phase peptide synthesis. In fact, amino acids bearing
acid-labile protecting groups on their side chains were incor-
porated into the synthetic strategy to demonstrate the possi-
bility of orthogonal-protection with the nosyl protecting
group.
3. Conclusions
In conclusion we describe a solution phase synthetic strategy
to prepare short peptide sequences using the nosyl group to
protect the amino function of a-amino acids under mild con-
ditions and in a rapid and efficient way.

A. Leggio et al. / Tetrahedron 63 (2007) 8164–8173
8169
The results obtained demonstrate that the nosyl group com-
bines very well with different side-chain protecting groups
widely used in solution phase peptide synthesis based on
the Fmoc strategy. Therefore, the nosyl group could be con-
sidered as an alternative to the Fmoc group in solution phase
peptide synthesis because its removal from the terminal ami-
no function of peptide chain provides the deprotected pep-
tide with high purity without using complex purification
procedures.
All reactions were monitored by thin-layer chromatography
using silica gel 60-F254 precoated glass plates. When
required, the reactions were carried out under an inert
atmosphere (N2).
4.2. General synthetic procedure for N-nosyl-dipeptides
4a and 4b
Mercaptoacetic acid (3 mmol) was added to a solution of 1a
and 1b (1 mmol) in dry acetonitrile (10 mL) under N2 and
stirred at reflux. Sodium methoxide (12 mmol) was then
added to the solution with a variable amount of methanol
to facilitate the sodium methoxide solubilization. The result-
ing mixture was stirred forw1 h monitoring the conversion
of 1a and 1b by TLC (diethyl ether/petroleum ether, 60:40
v/v). Then the solvent was evaporated under reduced pres-
sure and the residue acidified with 1 M HCl and extracted
with ethyl acetate (3ꢂ10 mL). The aqueous phase was basi-
fied with saturated aqueous Na2CO3. The basic liquors, con-
taining the N-deprotected amino acid methyl esters 2a and
2b, were then treated with a solution of N-nosyl-D-alanine
chloride 3 (1 mmol) in dry ethanol-free chloroform
(10 mL). The reaction mixture was stirred at room tempera-
ture forw1 h and the organic layer was separated. The aque-
ous phase was extracted with three additional portions of
chloroform (3ꢂ10 mL). The combined organic extracts
were washed with a 1 M aqueous solution of HCl and a sat-
urated aqueous solution of NaCl, dried (Na2SO4), and evap-
orated to dryness to afford the N-nosyl-tripeptides 4a and 4b
as white solids in 71–78% yields.
Furthermore, the reagent necessary to synthesize N-nosyl-
a-amino acids, nosyl chloride, is commercially available
and considerably cheaper than the reagent employed for
the introduction of the Fmoc protecting group, 9-fluorenyl-
methoxycarbonyloxy succinimide.8
The procedure developed is particularly convenient to syn-
thesize N-methylated peptide on specific amino acids be-
cause in this case it is possible to use the nosyl group as
the sole a-amino function protecting group.
The results shown here demonstrate that the developed
method based on the use of N-nosyl-a-amino acids could
provide new potential synthetic strategies for peptide
synthesis.
4. Experimental
4.1. General experimental procedures
All reagents were commercially obtained (Aldrich, Fluka) at
highest commercial quality and used without further purifi-
cation except where noted. Solvents were purified and dried
by standard procedures and distilled prior to use. Melting
points were recorded on a Kofler hot-stage apparatus and
4.2.1. N-Nosyl-D-alanyl-L-valine methyl ester 4a. The
product was prepared by general procedure A using 1a
(0.20 g, 0.63 mmol) in dry acetonitrile (10 mL), mercapto-
acetic acid (0.13 mL, 1.9 mmol), and sodium methoxide
(0.41 g, 7.56 mmol) in methanol (5 mL). The reaction was
stirred at reflux for 50 min. The afforded unmasked dipep-
tide in a 9% aqueous solution of NaHCO3 was treated with
3 (0.18 g, 0.63 mmol) in ethanol-free chloroform. The reac-
tion was stirred at room temperature for 45 min. The subse-
quent work up afforded 0.14 g of the title compound 4a as
a white solid (0.49 mmol, 78%): mp 146–147 ꢀC. [a]D20
ꢁ19.7 (c 0.62, CHCl3); IR (KBr): n 3350, 3119, 2976,
1
are uncorrected. The H NMR and 13C NMR spectra were
recorded at 300 and 75 MHz, respectively, using CDCl3 or
DMSO-d6 as solvents. Chemical shifts are reported in units
of parts per million and all coupling constants are reported in
Hertz. Optical rotations were measured on a Perkin–Elmer
polarimeter at 20 ꢀC. [a]D values are given in units of
10ꢁ1 deg cm2 gꢁ1
.
1
1730, 1645, 1536, 1352, 1042, 856, 742 cmꢁ1. H NMR
GC/MS analyses were performed using an HP-5MS
(30 mꢂ0.25 mm, PhMesiloxane 5%) capillary column.
The mass detector was operated in the electron impact ion-
ization mode (EI-MS) with an electron energy of 70 eV.
Mass spectra were recorded on a Vacuum Generators
ZAB-2F spectrometer, using 3-nitrobenzyl alcohol as ma-
trix, by fast atom bombardment (FAB+MS), with a neutral
xenon beam operating at 8 keV and a total current of
10 mA. The MALDI mass spectrum was acquired on
a 4700 proteomics analyzer mass spectrometer equipped
with 200 Hz, Nd:YAG laser at 355 nm wavelength. The
MS spectrum was acquired in reflectron mode (20 keV
accelerating voltage), with 400 ns delayed extraction,
averaging 2000 laser shots. a-Cyano-4-hydroxycinnamic
acid (HCCA) was used as matrix. 0.45 mL of a premixed
solution of HCCA and sample (800:1) dissolved in
MeOH/H20 (1:1) were spotted on the matrix target, dried
at room temperature, and analyzed with the mass
spectrometer.
(300 MHz, CDCl3): d 8.32 (2H, d, J¼8.6 Hz), 8.05 (2H, d,
J¼8.6 Hz), 6.51 (1H, d, J¼8.8 Hz), 5.98 (1H, d, J¼
6.7 Hz), 4.33 (1H, dd, J¼8.8, 4.7 Hz), 3.98 (1H, m), 3.73
(3H, s), 2.12 (1H, m), 1.37 (3H, d, J¼7.0 Hz), 0.87 (3H, d,
J¼6.8 Hz), 0.83 (3H, d, J¼6.8 Hz). 13C NMR (75 MHz,
CDCl3): d 172.09, 170.76, 150.12, 145.41, 128.53, 124.43,
56.94, 52.65, 52.48, 31.27, 20.33, 18.91, 17.47. MS (EI)
m/z (rel intensity %) 328 (M+ꢁCOOCH3ꢃ , 13), 229 (100),
186 (36), 158 (9), 130 (20), 122 (30), 72 (31). Anal. Calcd
for C15H21N3O7S: C, 46.50; H, 5.46; N, 10.85; S, 8.28.
Found: C, 46.31; H, 5.48; N, 10.88; S, 8.24.
4.2.2. N-Nosyl-D-alanyl-D-valine methyl ester 4b. The
product was prepared following the general procedure de-
scribed above using 1b (0.20 g, 0.63 mmol) in dry aceto-
nitrile (10 mL), mercaptoacetic acid (0.13 mL, 1.9 mmol),
and sodium methoxide (0.41 g, 7.56 mmol) in methanol
(5 mL). The reaction was stirred at reflux for 1 h. The af-
forded unmasked dipeptide in an aqueous 9% solution of

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A. Leggio et al. / Tetrahedron 63 (2007) 8164–8173
NaHCO3 was treated with 3 (0.18 g, 0.63 mmol) in ethanol-
free chloroform. The reaction was stirred at room tempera-
ture for 45 min. The subsequent work up afforded 0.13 g
of the title compound 4b as a white solid (0.44 mmol,
71%): mp 160–162 ꢀC. [a]D20 +36.2 (c 0.64, CHCl3); IR
(KBr): n 3350, 3119, 2976, 1730, 1645, 1536, 1352, 1042,
J¼7.2 Hz), 1.12 (1H, m), 0.80–1.00 (12H, m). 13C NMR
(75 MHz, DMSO-d6): d 172.52, 171.60, 169.81, 149.74,
146.26, 127.77, 124.11, 61.62. 56.85, 52.79, 48.36, 38.38,
31.74, 24.89, 19.15, 18.85, 17.59, 15.06, 11.09. MS (EI)
m/z (rel intensity %) 500 (Mꢃ+, 1%), 441 (4), 370 (3), 342
(13), 271 (16), 241 (22), 215 (25), 187 (100), 186 (13),
156 (16), 122 (13), 72 (41). Anal. Calcd for C21H32N4O8S:
C, 50.39; H, 6.44; N, 11.19; S, 6.41. Found: C, 50.54; H,
6.42; N, 11.29; S, 6.39.
1
856, 742 cmꢁ1. H NMR (300 MHz, CDCl3): d 8.32 (2H,
d, J¼8.6 Hz), 8.08 (2H, d, J¼8.6 Hz), 6.75 (1H, d,
J¼8.7 Hz), 6.50 (1H, d, J¼8.5 Hz), 4.38 (1H, dd, J¼8.7,
4.9 Hz), 4.06 (1H, m), 3.73 (3H, s), 2.02 (1H, m), 1.32
(3H, d, J¼7.0 Hz), 0.89 (3H, d, J¼6.9 Hz), 0.75 (3H, d,
J¼6.9 Hz). 13C NMR (75 MHz, CDCl3): d 172.06, 171.27,
149.98, 145.91, 128.34, 124.33, 57.25, 52.48, 52.38,
31.09, 19.76, 18.61, 17.54. MS (EI) m/z (rel intensity %)
328 (M+ꢁCOOCHꢃ3, 13), 229 (100), 186 (36), 158 (9), 130
(20), 122 (30), 72 (31). Anal. Calcd for C15H21N3O7S: C,
46.50; H, 5.46; N, 10.85; S, 8.28. Found: C, 46.68; H,
5.45; N, 10.86; S, 8.24.
4.3.2. N-Nosyl-L-isoleucyl-D-alanyl-D-valine methyl ester
7b. The product was prepared following the general proce-
dure described above using 4b (0.12 g, 0.31 mmol) in dry
acetonitrile (15 mL), mercaptoacetic acid (0.06 mL,
0.93 mmol), and sodium methoxide (0.27 g, 4.96 mmol) in
methanol (10 mL). The reaction was stirred at reflux for
2 h. The unmasked dipeptide in a 9% aqueous solution of
NaHCO3 was treated with 6 (0.05 g, 0.31 mmol) in etha-
nol-free chloroform. The reaction was stirred at room tem-
perature for 1 h. The subsequent work up afforded 0.13 g
of the title compound 7b as a white solid (0.26 mmol,
84%): mp 221–223 ꢀC. [a]D20 +31.7 (c 0.32, CHCl3); IR
(KBr): n 3378, 1637, 1545, 1361, 1352, 858, 800,
4.3. General synthetic procedure for N-nosyl-tripeptides
7a and 7b
Mercaptoacetic acid (3 mmol) was added to a solution of 4a
and 4b (1 mmol) in dry acetonitrile (10 mL) under N2 and
stirred at reflux. Solid sodium methoxide (16 mmol) was
then added to the solution with a variable amount of metha-
nol to facilitate the sodium methoxide solubilization. The
resulting mixture was stirred forw2 h monitoring the conver-
sion of 4a and 4b by TLC (diethyl ether/petroleum ether,
60:40 v/v). Then the solvent was evaporated under reduced
pressure and the residue acidified with 1 M HCl and ex-
tracted with ethyl acetate (3ꢂ10 mL). The aqueous phase
was basified with saturated aqueous Na2CO3. The basic
liquors, containing the N-deprotected amino acid methyl
esters 5a and 5b, were then treated with a solution of N-
nosyl-D-alanine chloride 6 (1 mmol) in dry ethanol-free
chloroform (10 mL). The reaction mixture was stirred at
room temperature forw1 h and the organic layer was sepa-
rated. The aqueous phase was extracted with three additional
portions of chloroform (3ꢂ10 mL). The combined organic
extracts were washed with a 1 M aqueous solution of HCl
and a saturated aqueous solution of NaCl, dried (Na2SO4),
and evaporated to dryness to afford the N-nosyl-tripeptides
7a and 7b as white solids in 79–92% yields.
1
741 cmꢁ1. H NMR (300 MHz, DMSO-d6): d 8.32 (2H, d,
J¼8.6 Hz), 8.14 (1H, d, J¼8.5 Hz), 8.04 (2H, d, J¼
8.6 Hz), 6.82 (1H, d, J¼7.3 Hz), 6.51 (1H, d, J¼8.2 Hz),
4.47 (1H, dd, J¼8.0, 6.6 Hz), 4.38 (1H, m), 3.55–3.70
(4H, m), 1.97 (1H, m), 1.42–1.62 (2H, m), 1.08 (1H, m),
0.93 (3H, d, J¼7.2 Hz), 0.73–0.86 (12H, m). 13C NMR
(75 MHz, DMSO-d6): d 172.56, 172.25, 169.57, 149.77,
147.22, 128.77, 124.48, 60.70, 57.72, 52.09, 47.91, 37.32,
30.30, 24.70, 19.25, 18.55, 18.15, 15.40, 10.87. MS (EI)
m/z (rel intensity %) 500 (Mꢃ+, 1%), 441 (2), 370 (3), 342
(14), 271 (15), 241 (30), 215 (23), 187 (100), 186 (12),
156 (26), 122 (12), 72 (40). Anal. Calcd for C21H32N4O8S:
C, 50.39; H, 6.44; N, 11.19; S, 6.41. Found: C, 50.54; H,
6.42; N, 11.21; S, 6.39.
4.4. General synthetic procedure for N-nosyl-amino
acids 9a–d
The side-chain protected a-amino acids 8a–d (1 mmol) were
dissolved in a dioxane/water solution and cooled to 0 ꢀC.
Dry triethylamine (20 mmol) and then a solution of p-nitro-
benzenesulfonyl chloride (1.5–1.6 mmol) in dioxane was
added slowly. The reaction mixture was stirred for 30–
50 min, monitoring the conversion of 8a–d by TLC (chloro-
form/methanol, 90:10 v/v). The solvent was removed under
reduced pressure and the residue basified with a 5% aqueous
solution of Na2CO3 and extracted with diethyl ether
(3ꢂ10 mL). The aqueous phase was acidified with a 5%
aqueous solution of KHSO4 (pH¼3–4) and extracted with
ethyl acetate. The organic layer was washed with water
and brine and then dried with Na2SO4. The solvent was
evaporated to afford the corresponding side-chain protected
N-nosyl amino acids 9a–d as white solids in 73–89% overall
yields.
4.3.1. N-Nosyl-L-isoleucyl-D-alanyl-L-valine methyl ester
7a. The product was prepared following the general proce-
dure described above using 4a (0.14 g, 0.36 mmol) in dry
acetonitrile (15 mL), mercaptoacetic acid (0.08 mL,
1.08 mmol), and sodium methoxide (0.31 g, 5.8 mmol) in
methanol (10 mL). The reaction was stirred at reflux for
2 h. The unmasked dipeptide in a 9% aqueous solution of
NaHCO3 was treated with 6 (0.12 g, 0.36 mmol) in etha-
nol-free chloroform. The reaction was stirred at room tem-
perature for 1 h. The subsequent work up afforded 0.16 g
of the title compound 7a as a white solid (0.32 mmol,
89%): mp 195–198 ꢀC. [a]D20 ꢁ24.5 (c 0.32, CHCl3); IR
(KBr): n 3376, 1637, 1545, 1361, 1352, 856, 800,
1
742 cmꢁ1. H NMR (300 MHz, DMSO-d6): d 8.22 (2H, d,
4.4.1. Na-Nosyl-N3-Boc-L-lysine 9a. The product was pre-
pared following the general procedure described above using
N3-Boc-L-lysine (8a) (0.4 g, 1.6 mmol) in dioxane/water
solution (20 mL), triethylamine (4.52 mL, 32 mmol), and
p-nitrobenzenesulfonyl chloride (0.57 g, 2.56 mmol). The
J¼8.6 Hz), 8.06 (2H, d, J¼8.6 Hz), 7.51 (1H, d, J¼8.4),
7.09 (1H, d, J¼7.8 Hz), 6.75 (1H, d, J¼9.0 Hz), 4.61 (1H,
dd, J¼8.0, 5.9 Hz), 4.19 (1H, m), 3.89 (3H, s), 3.71 (1H, t,
J¼8.1 Hz), 2.19 (1H, m), 1.58–1.78 (2H, m), 1.28 (3H, d,

A. Leggio et al. / Tetrahedron 63 (2007) 8164–8173
8171
reaction was stirred at room temperature for 30 min. The
subsequent work up afforded 0.51 g of the title compound
(1.18 mmol, 73%) as a white solid: mp 153–155 ꢀC. [a]D20
+20.2 (c 0.62, CH3OH); IR (KBr): n 3426, 3408, 3108,
136–138 ꢀC. [a]D20 ꢁ11.2 (c 0.60, CH3OH); IR (KBr): n
3426, 3358, 3112, 2964, 1725, 1616, 1533, 1348, 1184,
1112, 850, 743, 700. 1H NMR (300 MHz, CDCl3): d 12.81
(1H, br s), 8.15 (2H, d, J¼8.6 Hz), 7.90 (2H, d, J¼8.6 Hz),
7.28–7.02 (15H, m), 6.51 (1H, s), 6.00 (1H, br s), 4.02
(1H, m), 3.01 (1H, m), 2.83 (1H, m). 13C NMR (75 MHz,
CDCl3): d 34.15, 50.08, 72.77, 121.43, 126.33, 128.27,
128.32, 145.06, 145.81, 151.63, 173.78, 174.97. MS m/z
(%) 567.8831 [(M+Na)+, 100]. Anal. Calcd for
C29H25N3O7S: C, 62.24; H, 4.50; N, 7.51; S, 5.73. Found:
C, 62.11; H, 4.51; N, 7.48; S, 5.74.
1
2964, 1730, 1662, 1528, 1342, 1261, 1092, 800. H NMR
(300 MHz, DMSO-d6): d 12.72 (1H, br s), 8.58 (1H, d,
J¼8.7 Hz), 8.39 (2H, d, J¼8.4 Hz), 8.00 (2H, d, J¼
8.9 Hz), 6.75 (1H, m), 3.70 (1H, dd, J¼13.5, 8.4 Hz), 2.78
(2H, m), 1.40–1.60 (2H, m), 1.34 (9H, s), 1.10–1.27 (4H,
m). 13C NMR (75 MHz, DMSO-d6): d 172.99, 155.98,
149.85, 147.25, 144.76, 128.53, 124.79, 77.80, 56.10, 31.98,
29.20, 28.70, 22.76, 18.06. MS m/z (%) 453.8931 [(M+Na)+,
100]. Anal. Calcd for C17H25N3O8S: C, 47.32; H, 5.84; N,
9.74; S, 7.43. Found: C, 47.41; H, 5.86; N, 9.72; S, 7.40.
4.5. General synthetic procedure for side-chain
protected N-nosyl-dipeptides 11a–d
4.4.2. Na-Nosyl-O-tert-butyl-L-tyrosine 9b. The product
was prepared following the general procedure described
above using O-tert-butyl-L-tyrosine (8b) (0.4 g, 1.7 mmol)
in dioxane/water solution (20 mL), triethylamine (4.7 mL,
34 mmol), and p-nitrobenzenesulfonyl chloride (0.56 g,
2.6 mmol). The reaction was stirred at room temperature
for 50 min. The subsequent work up afforded 0.53 g of the
title compound (1.25 mmol, 75%) as a pale yellow solid:
mp 150–152 ꢀC. [a]D20 +1.9 (c 0.60, CH3OH); IR (KBr): n
3183, 2982, 1727, 1608, 1530, 1349, 1308, 1147, 850,
The a-amino acid methyl ester hydrochlorides 10a and 10b
(1 mmol), 1-hydroxybenzotriazole (1.1 mmol), N-methyl-
morpholine (1 mmol), and the side-chain protected Na-
nosyl-a-amino acids 9a–d (1 mmol) are dissolved in dry
tetrahydrofuran (20 mL). The solution was stirred and
cooled in an ice-water bath while dicyclohexylcarbodiimide
(1.15 mmol) is added. Stirring was continued for 1 h at 0 ꢀC
and an additional hour at room temperature monitoring the
reaction by TLC (chloroform/methanol, 80:20 v/v). N,N0-
Dicyclohexylurea which separated was removed by filtration
and the solvent evaporated in vacuo. A mixture of ethyl ace-
tate (30 mL) and a saturated solution of NaHCO3 in water
(10 mL) was added to the residue and the organic phase ex-
tracted with a 10% solution of citric acid in water (10 mL),
again with saturated NaHCO3 (10 mL) and brine. The solu-
tion was dried over Na2SO4, filtered, and evaporated to dry-
ness in vacuo to afford the side-chain N-nosyl-dipeptides
11a–d as pale yellow solids in 87–99% overall yields.
1
738. H NMR (300 MHz, DMSO-d6): d 12.76 (1H, br s),
8.70 (1H, br s), 8.20 (2H, d, J¼8.4 Hz), 7.75 (2H, d, J¼
8.9 Hz), 6.98 (2H, d, J¼8.2 Hz), 6.65 (2H, d, J¼8.2 Hz),
3.92 (1H, m), 2.92 (1H, m), 2.62 (2H, m), 1.37 (9H, s).
13C NMR (75 MHz, DMSO-d6): d 171.62, 151.69, 145.83,
145.83, 131.11, 128.45, 128.23, 121.46, 114. 35, 78.05,
54.78, 51.97, 36.13, 29.53. MS m/z (%) 444.8162 [(M+Na)+,
100]. Anal. Calcd for C19H22N2O7S: C, 54.02; H, 5.25; N,
6.63; O, 26.51; S, 7.59. Found: C, 53.80; H, 5.26; N, 6.62;
S, 7.61.
4.5.1. Na-Nosyl-N3-Boc-L-lysinyl-L-alanineOMe 11a. The
product was prepared following the general procedure de-
scribed above using L-alanine methyl ester hydrochloride
(10a) (0.1 g, 0.7 mmol), 1-hydroxybenzotriazole (0.1 g,
0.76 mmol), N-methylmorpholine (0.08 mL, 0.7 mmol),
Na-nosyl-N3-Boc-L-lysine (9a) (0.3 g, 0.7 mmol), and dicy-
clohexylcarbodiimide (0.16 g, 0.8 mmol) in dry tetrahydro-
furan (20 mL). The solution was stirred and cooled in an
ice-water bath for 1 h and then for an additional hour at
room temperature. The subsequent work up afforded 0.35 g
of the title compound (0.67 mmol, 90%) as a pale yellow
solid: mp 102–104 ꢀC. [a]D20 +15.6 (c 0.64, CHCl3); IR
(KBr): n 3336, 3260, 3108, 2931, 1741, 1686, 1647, 1529,
4.4.3. Na-Nosyl-S-trityl-L-cysteine 9c. The product was
prepared following the general procedure described above
using S-trityl-L-cysteine (8c) (0.5 g, 1.4 mmol) in dioxane/
water
solution
(20 mL),
triethylamine
(3.8 mL,
27.5 mmol), and p-nitrobenzenesulfonyl chloride (0.46 g,
2 mmol). The reaction was stirred at room temperature for
40 min. The subsequent work up afforded 0.63 g of the title
compound (1.15 mmol, 83%) as an orange solid: mp 99–
101 ꢀC. [a]D20 +19.2 (c 0.60, CH3OH); IR (KBr): n 3271,
3100, 3056, 1725, 1606, 1530, 1348, 1164, 1090, 854, 740,
1
700. H NMR (300 MHz, DMSO-d6) d 12.98 (1H, br s),
1
8.82 (2H, d, J¼8.4 Hz), 8.35 (2H, d, J¼8.4 Hz), 7.96 (1H,
d, J¼8.9 Hz), 7.16–7.34 (15H, m), 3.59 (1H, m), 2.32 (2H,
m). 13C NMR (75 MHz, DMSO-d6): d 174.90, 151.67,
145.83, 143.92, 129.38, 128.28, 128.21, 126.38, 121.44,
67.36, 55.74, 26.81. MS m/z (%) 571.9175 [(M+Na)+, 100].
Anal. Calcd for C28H24N2O6S2: C, 61.30; H, 4.41; N, 5.11;
S, 11.69. Found: C, 61.42; H, 4.42; N, 5.09; S, 11.64.
1450, 1352, 1261, 1166, 1090, 801. H NMR (300 MHz,
CDCl3): d 8.30 (2H, d, J¼8.8 Hz), 8.03 (2H, d, J¼8.7 Hz),
7.10 (1H, d, J¼8.1 Hz), 6.40 (1H, d, J¼5.5 Hz), 4.80 (1H,
m), 4.31 (1H, m), 3.85 (1H, m), 3.68 (3H, s), 3.08–3.18
(1H, m), 2.82–3.03 (1H, m), 1.65 (2H, m), 1.25–1.46 (3H,
m), 1.44 (9H, s), 1.06–1.17 (4H, m). 13C NMR (75 MHz,
CDCl3): d 172.84, 170.37, 156.74, 156.05, 150.05, 128.64,
124.22, 79.63, 56.33, 52.60, 49.31, 48.09, 38.98, 31.95,
29.01, 28.45, 24.90, 21.22, 17.90. MS m/z (%) 538.8431
[(M+Na)+, 100]. Anal. Calcd for C21H32N4O9S: C, 48.83;
H, 6.24; N, 10.85; S, 6.21. Found: C, 48.97; H, 6.23; N,
10.87; S, 6.18.
4.4.4. Na-Nosyl-Nb-trityl-L-asparagine 9d. The product
was prepared following the general procedure described
above using Nb-trityl-L-asparagine (8d) (0.3 g, 0.8 mmol)
in dioxane/water solution (20 mL), triethylamine
(2.23 mL, 16 mmol), and p-nitrobenzenesulfonyl chloride
(0.27 g, 1.2 mmol). The reaction was stirred at room temper-
ature for 40 min. The subsequent work up afforded 0.39 g of
the title compound (0.7 mmol, 89%) as a white solid: mp
4.5.2. Na-Nosyl-O-tert-butyl-L-tyrosinyl-L-valineOMe
11b. The product was prepared following the general pro-
cedure described above using L-valine methyl ester

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A. Leggio et al. / Tetrahedron 63 (2007) 8164–8173
hydrochloride (10b) (0.12 g, 0.71 mmol), 1-hydroxy-
benzotriazole (0.1 g, 0.78 mmol), N-methylmorpholine
(0.078 mL, 0.71 mmol), Na-nosyl-O-tert-butyl-L-tyrosine
(9b) (0.3 g, 0.71 mmol), and dicyclohexylcarbodiimide
(0.168 g, 0.82 mmol) in dry tetrahydrofuran (20 mL). The
solution was stirred and cooled in an ice-water bath for 1 h
and then for an additional hour at room temperature. The
subsequent work up afforded 0.31 g of the title compound
(0.58 mmol, 82%) as a pale yellow solid: mp 64–66 ꢀC.
[a]2D0 ꢁ12.1 (c 0.58, CHCl3); IR (KBr): n 3394, 3213,
3119, 2963, 1749, 1637, 1535, 1351, 1261, 1093, 1024,
799. 1H NMR (300 MHz, CDCl3): d 8.20 (2H, d,
J¼8.9 Hz), 7.78 (2H, d, J¼8.4 Hz), 6.91 (2H, d, J¼
8.5 Hz), 6.71 (2H, d, J¼8.5 Hz), 6.62 (1H, d, J¼8.2 Hz),
6.05 (1H, d, J¼8.5 Hz), 4.38 (1H, dd, J¼8.5, 4.8 Hz), 4.00
(1H, m), 3.72 (3H, s), 3.02 (1H, dd, J¼14.1, 8.3 Hz), 2.88
(1H, dd, J¼14.1, 5.9 Hz), 2.08 (1H, m), 1.30 (9H, s), 0.81
(6H, d, J¼6.9 Hz). 13C NMR (75 MHz, CDCl3): d 171.67,
170.07, 154.87, 149.94, 145.27, 129.83, 129.70, 128.16,
124.33, 124.29, 58.70, 57.46, 52.35, 38.17, 33.83, 31.24,
28.74, 18.85, 17.70. MS m/z (%) 557.9979 [(M+Na)+,
100]. Anal. Calcd for C25H33N3O8S: C, 56.06; H, 6.21; N,
7.85; S, 5.99. Found: C, 55.83; H, 6.19; N, 7.88; S, 6.00.
2932, 2849, 1742, 1666, 1625, 1531, 1348, 1165, 1089, 853,
700. 1H NMR (300 MHz, CDCl3): d 8.15 (2H, d, J¼8.8 Hz),
7.90 (2H, d, J¼8.9 Hz), 7.10–7.28 (15H, m), 6.53 (1H, s),
5.98 (1H, br s,), 5.56 (1H, d, J¼9.8 Hz), 4.32 (1H, m),
4.05 (1H, m), 3.68 (3H, s), 3.09 (1H, m), 2.84 (1H, m),
1.25 (3H, d, J¼7.2 Hz). 13C NMR (75 MHz, CDCl3):
d 172.28, 169.98, 169.09, 150.04, 145.46, 143.80, 128.45,
128.30, 127.91, 127.13, 124.34, 70.90, 52.90, 52.35,
48.40, 33.70, 17.36. MS m/z (%) 652.7740 [(M+Na)+,
100]. Anal. Calcd for C33H32N4O8S: C, 61.48; H, 5.00; N,
8.69; S, 4.97. Found: C, 61.43; H, 4.98; N, 8.71; S, 4.96.
4.6. General synthetic procedure for side-chain
protected N-nosyl-tripeptides 14a–c
Mercaptoacetic acid (3 mmol) was added to a solution of
11a–c (1 mmol) in dry acetonitrile (10 mL) under N2 and
stirred at reflux. Sodium methoxide (16 mmol) was then
added to the solution with a variable amount of methanol to
facilitate the sodium methoxide solubilization. The resulting
mixture was stirred for 1–2 h monitoring the conversion of
11a–c by TLC (diethyl ether/petroleum ether, 90:10 v/v).
Then the solvent was evaporated under reduced pressure
and the residue acidified with a 5% solution of KHSO4 in wa-
ter and extracted with ethyl acetate (3ꢂ10 mL). The aqueous
phase was basified with a 5% solution of Na2CO3 in water.
The basic liquors, containing the N-deprotected amino acid
methyl esters 12a–c, were then treated with a solution of
N-nosyl-a-amino acid chloride 13, 6 (1 mmol) in dry etha-
nol-free chloroform (5 mL). The reaction mixture was stirred
at room temperature forw2 h and the organic layer was sep-
arated. The aqueous phase was extracted with three addi-
tional portions of chloroform (3ꢂ10 mL). The combined
organic extracts were washed with a 5% aqueous solution
of citric acid and a saturated aqueous solution of NaCl, dried
(Na2SO4), and evaporated to dryness to afford the N-nosyl-
tripeptides 14a–c as white solids in 71–85% yields.
4.5.3. Na-Nosyl-S-trityl-L-cysteinyl-L-alanineOMe 11c.
The product was prepared following the general proce-
dure described above using L-alanine methyl ester
hydrochloride (10a) (0.076 g, 0.55 mmol), 1-hydroxybenzo-
triazole (0.08 g, 0.6 mmol), N-methylmorpholine (0.06 mL,
0.55 mmol), Na,Na-nosyl-S-trityl-L-cysteine (9c) (0.3 g,
0.55 mmol), and dicyclohexylcarbodiimide (0.13 g,
0.63 mmol) in dry tetrahydrofuran (20 mL). The solution
was stirred and cooled in an ice-water bath for 1 h and
then for an additional hour at room temperature. The sub-
sequent work up afforded 0.3 g of the title compound
(0.47 mmol, 87%) as a pale yellow solid: mp 138–142 ꢀC.
[a]2D0 +21.9 (c 0.64, CHCl3); IR (KBr): n 3332, 3274,
3059, 2932, 1731, 1663, 1536, 1347, 1261, 1164, 1089,
800, 698. 1H NMR (300 MHz, CDCl3): d 8.18 (2H, d,
J¼8.8 Hz), 7.92 (2H, d, J¼8.7 Hz), 7.28 (15H, m), 6.20
(1H, d, J¼7.2 Hz), 5.70 (1H, d, J¼7.5 Hz), 4.32 (1H, m),
3.68 (3H, s), 3.14 (1H, m), 2.60 (1H, dd, J¼13.8, 5.4 Hz),
2.48 (1H, dd, J¼13.8, 8.4 Hz), 1.22 (3H, d, J¼7.2 Hz).
13C NMR (75 MHz, CDCl3): d 172.25, 168.17, 149.90,
145.35, 143.90, 129.32, 128.57, 127.80, 126.98, 123.91,
67.30, 56.03, 52.43, 48.22, 24.77, 17.92. MS m/z (%)
656.9899 [(M+Na)+, 100]. Anal. Calcd for C32H31N3O7S2:
C, 60.65; H, 4.93; N, 6.63; S, 10.12. Found: C, 60.83; H,
4.92; N, 6.64; S, 10.08.
4.6.1. Na-Nosyl-L-valyl-N3-Boc-L-lysinyl-L-alanineOMe
14a. The product was prepared following the general proce-
dure described above using 11a (0.17 g, 0.33 mmol) in dry
acetonitrile (10 mL), mercaptoacetic acid (0.07 mL,
0.99 mmol), and sodium methoxide (0.29 g, 5.3 mmol) in
methanol (10 mL). The reaction was stirred at reflux for
1 h. The afforded unmasked dipeptide in a 5% aqueous solu-
tion of Na2CO3 was treated with Na-nosyl-L-valine chloride
(13) (0.105 g, 0.33 mmol) in ethanol-free chloroform. The
reaction was stirred at room temperature for 30 min. The sub-
sequent work up afforded 0.17 g of the title compound 14a as
a white solid (0.27 mmol, 85%): mp 166–168 ꢀC. [a]D20
ꢁ11.7 (c 0.60, CHCl3); IR (KBr): n 3315, 3260, 3103,
2964, 2937, 1727, 1686, 1633, 1531, 1349, 1261, 1170,
4.5.4. Na-Nosyl-Nb-trityl-L-asparaginyl-L-alanineOMe
11d. The product was prepared following the general proce-
dure described above using L-alanine methyl ester
hydrochloride (10a) (0.051 g, 0.37 mmol), 1-hydroxybenzo-
1
1091, 798. H NMR (300 MHz, DMSO-d6): d 8.31 (2H, d,
J¼7.5 Hz), 8.08 (2H, d, J¼7.8 Hz), 7.15–7.28 (2H, m),
6.51 (1H, m), 4.90 (1H, m), 4.45–4.54 (2H, m), 3.65–3.80
(4H, m), 3.08 (2H, m), 2.18 (1H, m), 1.65 (2H, m), 1.45
(9H, s), 1.36 (3H, d, J¼6.7 Hz), 1.10–1.27 (4H, m), 0.85
(6H, m). 13C NMR (75 MHz, DMSO-d6): d 173.00, 171.00,
170.47, 156.50, 149.23, 146.10, 128.57, 124.18, 62.21,
52.61, 48.22, 39.72, 32.05, 31.84, 29.71, 29.37, 28.45, 22.15,
19.17, 17.79, 17.66. MS m/z (%) 637.9419 [(M+Na)+]. Anal.
Calcd for C26H41N5O10S: C, 50.72; H, 6.71; N, 11.37; S,
5.21. Found: C, 50.61; H, 6.72; N, 11.33; S, 5.22.
triazole
(0.055 g,
0.4 mmol),
N-methylmorpholine
(0.04 mL, 0.37 mmol), Na-nosyl-Nb-trityl-L-asparagine (9d)
(0.2 g, 0.37 mmol), and dicyclohexylcarbodiimide (0.08 g,
0.42 mmol) in dry tetrahydrofuran (20 mL). The solution
was stirred and cooled in an ice-water bath for 1 h and
then for an additional hour at room temperature. The sub-
sequent work up afforded 0.22 g of the title compound
(0.35 mmol, 95%) as a pale yellow solid: mp 138–141 ꢀC.
[a]2D0 +36.6 (c 0.56, CHCl3); IR (KBr): n 3325, 3062,

A. Leggio et al. / Tetrahedron 63 (2007) 8164–8173
8173
4.6.2. Na-Nosyl-L-leucyl-O-tert-butyl-L-tyrosinyl-L-valine
OMe 14b. The product was prepared following the gen-
eral procedure described above using 11b (0.20 g,
0.37 mmol) in dry acetonitrile (10 mL), mercaptoacetic
acid (0.08 mL, 1.12 mmol), and sodium methoxide (0.32 g,
5.9 mmol) in methanol (10 mL). The reaction was stirred at
reflux for 2 h. The afforded unmasked dipeptide in an aque-
ous 5% solution of Na2CO3 was treated with Na-nosyl-L-iso-
leucine chloride (6) (0.12 g, 0.37 mmol) in ethanol-free
chloroform. The reaction was stirred at room temperature
for 1 h. The subsequent work up afforded 0.18 g of the title
compound 14b as a white solid (0.27 mmol, 75%): mp 203–
205 ꢀC. [a]D20 ꢁ2.6 (c 0.58, CHCl3); IR (KBr): n 3324, 3270,
3177, 2963, 1743, 1644, 1530, 1261, 1165, 1094, 1024, 800.
1H NMR (300 MHz, CDCl3): d 8.31 (2H, d, J¼8.6 Hz), 8.04
(2H, d, J¼8.6 Hz), 6.90–7.08 (5H, m), 6.50 (1H, d,
J¼8.4 Hz), 6.18 (1H, m), 4.61 (1H, m), 4.39 (1H, m), 3.88
(1H, m), 3.71 (3H, s), 2.85 (2H, m), 2.05 (2H, m), 1.39–
1.50 (2H, m), 1.32 (9H, s), 0.70–0.87 (12H, m). 13C NMR
(75 MHz, CDCl3): d 171.58, 171.03, 170.59, 154.60,
150.07, 145.62, 129.69, 128.57, 124.47, 124.32, 57.63,
55.67, 54.56, 52.34, 42.34, 37.69, 30.97, 28.81, 24.35, 22.85,
21.19, 18.81, 17.89. MS m/z (%) 670.9496 [(M+Na)+, 100].
Anal. Calcd for C31H44N4O9S: C, 57.39; H, 6.84; N, 8.64; S,
4.94. Found: C, 57.50; H, 6.83; N, 8.66; S, 4.92.
128.41, 128.22, 127.23, 126.32, 124.07, 66.92, 61.96,
61.07, 51.92, 48.46, 31.47, 28.63, 18.85, 17.05, 13.88. MS
m/z (%) 755.8488 [(M+Na)+, 100]. Anal. Calcd for
C37H40N4O8S2: C, 60.64; H, 5.50; N, 7.64; S, 8.75. Found:
C, 60.52; H, 5.49; N, 7.66; S, 8.72.
References and notes
1. (a) Methods in Molecular Biology; Pennington, M. W., Dunn,
B. M., Eds.; Peptide and Protocols; Humana: Totowa, NJ,
1994; Vol. 35, pp 91–169; (b) Atherton, E.; Sheppard, R. C.
Solid Phase Peptide Synthesis: A Practical Approach; Oxford-
IRL: New York, NY, 1989.
2. Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic
Synthesis, 3rd ed.; Wiley: New York, NY, 1999.
3. (a) Carpino, L. A. Acc. Chem. Res. 1987, 20, 401–403; (b)
Atherton, E.; Sheppard, R. C. The Peptides; Undenfriend, S.,
Meienhofer, J., Eds.; Academic: New York, NY, 1987; Vol. 9, p 1.
4. Sheppard, R. J. Pept. Sci. 2003, 9, 545–552.
5. (a) Carpino, L. A.; Cohen, B. J.; Stephens, K. E.; Sadat-Aalaee,
S. Y.; Tien, J. H.; Langridge, D. C. J. Org. Chem. 1986, 51,
3732–3734; (b) Beyermann, M.; Bienert, M.; Niedrich, H.
J. Org. Chem. 1990, 55, 721–728.
6. (a) Di Gioia, M. L.; Leggio, A.; Le Pera, A.; Liguori, A.; Napoli,
A.; Siciliano, C.; Sindona, G. J. Org. Chem. 2003, 68, 7416–
7421; (b) Di Gioia, M. L.; Leggio, A.; Liguori, A. J. Org.
Chem. 2005, 70, 3892–3897; (c) Fukuyama, T.; Jow, C.-K.;
Cheung, M. Tetrahedron Lett. 1995, 36, 6373–6374; (d)
Miller, S. C.; Scanlan, T. S. J. Am. Chem. Soc. 1997, 119,
2301–2302; (e) Fukuyama, T.; Cheung, M.; Jow, C.-K.; Aidai,
Y. Tetrahedron Lett. 1997, 38, 5831–5834; (f) Miller, S. C.;
Scanlan, T. S. J. Am. Chem. Soc. 1998, 120, 2690–2691; (g)
Reichwein, J. F.; Liskamp, R. M. Tetrahedron Lett. 1998, 39,
1243–1246; (h) Narayan, R. S.; VanNieuwenhze, M. S. Org.
Lett. 2005, 7, 2655–2658; (i) Ferraris, D.; Dudding, T.; Young,
B.; Drury, W. J., III; Lactka, T. J. Org. Chem. 1999, 64, 2168–
2169; (l) Christensen, C.; Rasmus, P. C.; Begtrup, M.;
Kristensen, J. L. Tetrahedron Lett. 2004, 45, 7991–7993; (m)
Krois, D.; Simonetti, M.; Wunsch, E. Monatsh. Chem. 1989,
120, 1029–1041.
4.6.3. Na-Nosyl-L-valyl-S-trityl-L-cysteinyl-L-alanine
OMe 14c. The product was prepared following the gen-
eral procedure described above using 11c (0.13 g,
0.25 mmol) in dry acetonitrile (10 mL), mercaptoacetic
acid (0.052 mL, 0.75 mmol), and sodium methoxide
(0.22 g, 4.0 mmol) in methanol (10 mL). The reaction was
stirred at reflux for 90 min. The afforded unmasked dipep-
tide in a 5% aqueous solution of Na2CO3 was treated with
0.08 g (0.25 mmol) Na-nosyl-L-valine chloride (13) in etha-
nol-free chloroform. The reaction was stirred at room tem-
perature for 1 h. The subsequent work up afforded 0.13 g
of the title compound 14c as a yellow solid (0.18 mmol,
71%): mp 180–182 ꢀC. IR (KBr): n 3338, 3261, 2963,
1
1733, 1643, 1529, 1350, 1265, 1087, 1030, 806, 697. H
NMR (300 MHz, CDCl3): d 8.35 (2H, d, J¼9.0 Hz), 8.05
(2H, d, J¼9.0 Hz), 7.28 (15H, m), 6.20 (1H, d, J¼7.2 Hz),
5.70 (1H, d, J¼7.5 Hz), 5.56 (1H, d, J¼9.8 Hz), 4.32 (1H,
m), 3.95 (1H, m), 3.55 (3H, s), 3.14 (1H, m), 2.58–2.70
(2H, m), 2.11 (1H, m), 1.22 (3H, d, J¼7.2 Hz), 0.98 (3H,
d, J¼6.8 Hz), 0.88 (3H, d, J¼6.8 Hz). 13C NMR (75 MHz,
CDCl3): d 171.84, 171.63, 171.17, 151.62, 145.83, 143.95,
7. Methyl 2-(4-nitrophenylthio)acetate: MS (EI) m/z (rel intensity
%) 227 (M+, 82), 168 (100), 151 (8), 137 (6), 122 (56), 121
(58).
8. Based on the Aldrich 2007/08 catalog, where 4-nitrobenzenesul-
fonyl chloride is V 269/mol and 9-fluorenylmethyloxycarbony-
loxy succinimide is V 3305/mol.