The dimethylsulfoxonium methylide as unique reagent for the simultaneous deprotection of amino and carboxyl function of N-Fmoc-α-amino acid and N-Fmoc-peptide esters

Article abstract of DOI:10.1016/j.tet.2012.12.052

The dimethylsulfoxonium methylide is described as a unique and useful reagent for the simultaneous deprotection of amino and carboxyl function of N-Fmoc-α-amino acid and N-Fmoc-peptide esters. The new methodology was applied successfully both to solution- and solid-phase peptide synthesis. The adopted methodology was extended successfully also to peptides containing amino acids bearing acid-sensitive protecting group in side chains. Furthermore no measurable epimerization was observed in the deprotection reaction of N-Fmoc-dipeptide methyl esters with dimethylsulfoxonium methylide.

Full text of DOI:10.1016/j.tet.2012.12.052

Tetrahedron 69 (2013) 2010e2016
Contents lists available at SciVerse ScienceDirect
Tetrahedron
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The dimethylsulfoxonium methylide as unique reagent for the
simultaneous deprotection of amino and carboxyl function
of N-Fmoc-a-amino acid and N-Fmoc-peptide esters
*
Mariagiovanna Spinella, Rosaria De Marco, Emilia L. Belsito, Antonella Leggio ,
*
Angelo Liguori
ꢀ
Dipartimento di Scienze Farmaceutiche, Universita della Calabria, Ed. Polifunzionale, I-87036 Arcavacata di Rende (CS), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The dimethylsulfoxonium methylide is described as a unique and useful reagent for the simultaneous
Received 11 September 2012
Received in revised form 10 December 2012
Accepted 21 December 2012
Available online 2 January 2013
deprotection of amino and carboxyl function of N-Fmoc-a-amino acid and N-Fmoc-peptide esters. The
new methodology was applied successfully both to solution- and solid-phase peptide synthesis. The
adopted methodology was extended successfully also to peptides containing amino acids bearing acid-
sensitive protecting group in side chains. Furthermore no measurable epimerization was observed in
the deprotection reaction of N-Fmoc-dipeptide methyl esters with dimethylsulfoxonium methylide.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Dimethylsulfoxonium methylide
Ester cleavage
Fmoc deprotection
Wang resin
Peptides
1. Introduction
We have recently developed a new methodology for the
deprotection of ester function by using dimethylsulfoxonium
One of the most widely used strategies in peptide synthesis is
based on the masking of the amino function of -amino acids by the
methylide.7 The treatment of methyl esters with dimethysulfoxo-
nium methylide leads to efficient cleavage of ester function and
the obtainment of the corresponding carboxyl acids. This procedure
is fast and proceeds under mild conditions also with complex
esters.
a
9-fluorenylmethoxycarbonyl (Fmoc) group.1 This approach has
found extended applications due to many advantages presented by
the Fmoc protecting group. It is stable under various reaction
conditions such as oxidation and reduction during the course of
multi-step synthesis or total synthesis of natural products.2 In
addition it shows high stability towards trifluoroacetic acid
(CF3COOH), hydrogen bromide (HBr), which on the contrary,
removes Boc (ter-butoxycarbonyl) and Cbz (benzyloxycarbonyl)3
groups, and tertiary amines. The Fmoc group is easily and typi-
The reaction has been utilized to convert N-nosyl-, N-Cbz- and
N-Boc-protected amino acid methyl esters into the corresponding
amino acids free on the carboxyl function. The deprotection re-
action of carboxyl function was fast in all cases and produced the
corresponding acids in high yields also in the case of N -3 Boc lysine,
S-trityl cysteine and O-benzyl-L-tyrosine. The above confirmed the
cally removed by treatment of N-Fmoc-
a
-amino acids with a large
applicability of the procedure also in presence of acid-sensitive
protecting groups in side chain and on amino function. Therefore,
the investigation of the stability of Fmoc group on the amino
excess of 20% piperidine solution in DMF.4 However, some limita-
tions exist when this procedure is applied to solution-phase pep-
tide synthesis5 due to the low volatility of these solvents and the
formation of the highly reactive dibenzofulvene (DBF) that induces
various side reactions (polymerization, addition of amines and so
on) causing difficulties in the purification step of the deprotected
product. The sensitivity of the Fmoc group to Lewis acid is also
reported.6
function of
thylsulfoxonium methylide seemed to us of particular interest.
During the course of our study, we found that N-Fmoc- -amino
a-amino acid methyl esters in presence of dime-
a
acid methyl esters, treated under the same conditions used with
N-nosyl-, N-Cbz- and N-Boc-amino acid1 methyl esters, did not
provide any product with structure comparable to the corre-
sponding N-Fmoc-a-amino acids free on carboxyl function. This
result prompted us to investigate extensively the reaction progress
of N-Fmoc-a-amino acid methyl esters with dimethylsulfoxonium
methylide.
* Corresponding authors. Tel.: þ39 (0)984 493199; fax: þ39 (0)984 493265
(A.Le.); tel.: þ39 (0)984 493205; fax: þ39 (0)984 493265 (A.Li.); e-mail addresses:
a.leggio@unical.it (A. Leggio), A.Liguori@unical.it (A. Liguori).
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.12.052

M. Spinella et al. / Tetrahedron 69 (2013) 2010e2016
2011
2. Results and discussions
In this work we studied the reactivity of
a
-amino acid methyl
esters protected on amino function with 9-fluorenylmethoxy
carbonyl group (Fmoc) with dimethylsulfoxonium methylide. In
a preliminary experiment N-Fmoc- -alanine methyl ester (1a)
L
(1 mmol) was treated at room temperature with dimethylsulfoxo-
nium methylide (2) (2 mmol), freshly prepared from trime-
thylsulfoxonium iodide and sodium hydride. The reaction was
monitored by thin-layer chromatography (TLC) and the conversion
of the starting material was completed in 20 min. TLC analysis of
the reaction mixture clearly revealed the formation of a new
product with a low Rf value, which gave a positive ninhydrin re-
action. The observed product probably should have the amino
9.0 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
function unmasked. The hypothesis that it was the -alanine (3a)
L
(Scheme 1), deprotected on both amino and carboxyl function,
prompted us to recover the product after derivatization of amino
function. To this aim, the crude reaction mixture, after hydrolysis
and elimination of DBF by simple extraction with diethyl ether, was
treated with nosyl chloride under basic conditions to obtain the
corresponding N-nosyl derivative 4a (Scheme 1).
Fig. 1. 1H NMR (CDCl3) analysis of the N-nosyl-L-isoleucine (4e).
peptide methyl esters. The application of this methodology to N-
Fmoc-peptide methyl esters required the preliminary investigation
of the stability of the amidic function towards the dime-
thylsulfoxonium methylide (2). To this aim, the N-Fmoc-L-valine
benzylamide (5), chosen as model system, was synthetized9 and
treated with the ylide 2 under the usually adopted conditions
(Scheme 2).
Scheme 1. Reaction of N-Fmoc-protected
methylsulfoxonium methylide (2).
a-amino acid methyl esters 1aee with di-
The 1H NMR spectrum of the reaction product, recovered after
acidic work up, clearly showed the presence of only N-nosyl- -al-
anine (4a).8 This result demonstrated that the reaction of N-Fmoc-
-alanine methyl ester (1a) (Scheme 1, Table 1) with the ylide 2
provided the corresponding amino acid -alanine 3a free on both
amino and carboxyl functions. Encouraged by this result, the
methodology was applied to other -amino acid methyl esters
N-Fmoc protected 1bee (Scheme 1, Table 1). The corresponding N-
Scheme 2. Reaction of N-Fmoc-L-valine benzylamide (5) with the dimethylsulfoxo-
nium methylide (2).
L
L
After a simple work up, the -valine benzylamide (7) was re-
L
L
covered as unique reaction product in 90% yield. This result dem-
onstrated that the amidic function is not affected by the
dimethylsulfoxonium methylide (2). The deprotection methodol-
a
nosyl-a
-amino acids 4bee8 were obtained in high yields (87e90%).
ogy was then applied to N-Fmoc-L-alanyl-L-alanine methyl ester
(8f) (Scheme 3). The treatment of dipeptide 8f with the ylide 2 gave
the corresponding dipeptide 9f free on both amino and carboxyl
function after only 20 min. The product 9f was recovered as
N-acetyl derivative 10f after treatment of the hydrolyzed reaction
mixture with acetic anhydride (Scheme 3).
Table 1
Results of the reaction of N-Fmoc-
a
-amino acid methyl esters 1aee with dime-
thylsulfoxonium methylide (2)
R
Yield (%)
90
4a
4b
4c
4d
4e
CH3
CH2CH(CH3)2
CH(CH3)2
CH2Ph
87
88
88
87
CH(CH3)CH2 CH3
It is worth noting that the removal of the Fmoc group from the
N-protected- -amino acid derivatives 1aee occurred without evi-
dence of racemization at the chiral -carbon atoms. This aspect was
a
a
investigated applying the reaction to N-Fmoc-L-isoleucine methyl
ester (1e). In fact, the 1H NMR analysis of the crude reaction product
4e did not show any signals relative to a possible epimer resulting
from an inversion of the configuration at the
a carbon atom (Fig. 1).
Another interesting goal of our study was the extension of the
above described deprotection procedure to N-Fmoc protected
Scheme 3. Reaction of N-Fmoc-dipeptide methyl esters 8feg with dimethylsulfox-
oium methylide (2).

2012
M. Spinella et al. / Tetrahedron 69 (2013) 2010e2016
The stereochemistry of the reaction was also investigated by
performing the deprotection reaction on N-Fmoc- -alanyl- -ala-
nine methyl ester (8g, Scheme 3), the diastereoisomer of 8f in order
to obtain N-acetyl- -alanyl- -alanine (10g), the diastereoisomer of
D
L
D
L
10f (Table 2). The crude N-acetylated dipeptides (10f and 10g) were
analyzed by 1H NMR. The corresponding spectra (a and b, Fig. 2)
revealed the presence of signals corresponding to only one di-
astereoisomer in each spectrum. To confirm this result a mixture of
10f (10 mg) and 10g (10 mg) was also analyzed by 1H NMR. The 1H
NMR spectrum of the mixture (c, Fig. 2) showed the separation of
the singlets at 1.81 and 1.82 ppm relative to the methyl protons of
acetyl group of the two diastereoisomers and the separation of the
doublets at 1.24 and 1.26 ppm relative to the protons of one of the
side chain methyl group of the two diastereoisomers. On the con-
trary, in the 1H NMR spectrum of each diastereoisomer 10f and 10g
(Fig. 2) we observed only one peak at 1.26 and 1.81 ppm, in the case
of 10f and at 1.24 and 1.82 in the case of 10g.
Scheme 4. Reaction of N-Fmoc-tripeptide methyl ester 11 with dimethylsulfoxoium
methylide (2).
Table 2
Results of the reaction of N-Fmoc-dipeptide methyl esters 8feg with dime-
thylsulfoxoium methylide (2)
R
R1
Yield (%)
Scheme 5. Reaction of N-nosyl-L-Ala Wang resin 14 with dimethylsulfoxonium
methylide (2).
10f
10g
CH3
H
H
CH3
90
88
The nosyl protection on the amino function of 14 was chosen
because it results stable under the deprotection conditions1 and
consequently allows to characterize easily and quickly the reaction
product. After simple filtration and acidic hydrolysis the N-nosyl-L-
alanine (4a) was recovered in 80% yield (Scheme 5). Subsequently
an N-Fmoc protected dipeptide anchored by carboxyl function to
the Wang resin, the N-Fmoc-L-alanyl-L-valine Wang resin (15), was
treated with dimethylsulfoxonium methylide (2) in a molar ratio
1:2 with respect to the total active groups of the resin, assuming
that the loading of the Wang resin with the first amino acid, L-al-
anine, was quantitative (Scheme 6).
Fig. 2. (a) 1H NMR (DMSO-d6) analysis of 10f; (b) 1H NMR (DMSO-d6) analysis of 10g;
(c) 1H NMR (DMSO-d6) analysis of the mixture of 10f and 10g.
Scheme 6. Reaction of N-Fmoc-L-alanyl-L-valine Wang resin (15) with dime-
thylsulfoxonium methylide (2).
These data clearly indicate that the deprotection reaction pro-
ceeds without epimerization at the chiral centres present on the
starting substrate. The simultaneous deprotection of both amino
and carboxyl functions was tested also on a tripeptide system, the
After 30 min the reaction mixture was hydrolyzed and filtered.
The filtrate was treated with acetic anhydride under basic condi-
tions in order to convert the deprotected product 16 into the
corresponding N-acetyl derivative 17 that was recovered in 90%
yield. To confirm this result, also a tripeptide system containing an
N-Fmoc-L-leucyl-L-phenylalanyl-L-alanine methyl ester (11, Scheme
4), obtained by automated synthesizer and subsequently methyl-
ated on carboxyl function with a diethyl ether solution of diazo-
methane 0.66 M.
acid-labile side chain protecting group, the N-Fmoc-
L-valyl-L-O-
The conversion of 11 into the total deprotected tripeptide was
completed in 25 min. The deprotected product 12 was recovered as
N-nosyl derivative 13 (Scheme 4). All these successful results sug-
gested us to apply the reaction also to resin anchored peptide
systems. In particular, the methodology could represent a useful
procedure for cleavage of peptide from solid support, when the
peptide is anchored to the resin through an ester bond. In a pre-
tert-butyl-serinyl- -alanine Wang resin (18), was treated with the
L
dimethylsulfoxonium methylide (2) in 1:2 M ratio with respect to
the total active groups of the resin (Scheme 7).
The conversion of 18 into the deprotected product 19 was com-
plete in 30 min. Compound 19 was recovered as nosyl derivative 20
in 80% yield, keeping unchanged the acid-sensitive protecting group
on the side chain. All these experiments demonstrated that dime-
thylsulfoxonium methylide is an efficient reagent to deprotect in
liminary experiment the N-nosyl-
L
-alanine Wang resin 14 was
shaken with methylide 2 in THF (Scheme 5).

M. Spinella et al. / Tetrahedron 69 (2013) 2010e2016
2013
with CDCl3 and DMSO-d6 as solvents. Commercially available re-
agents were purchased from Aldrich Chemical Co. (Milan, Italy). The
dimethylsulfoxonium methylide was prepared following the pro-
cedure already reported in the literature.1 All reactions were carried
out under inert atmosphere (N2). The peptides, N-Fmoc-
phenylalanyl- -alanine, N-Fmoc- -alanyl- -valine Wang resin and N-
Fmoc- -valyl-O-tert-Butyl- -serinyl- -alanine Wang resin, were syn-
L-leucyl-L-
L
L
L
L
L
L
thetized by solid-phase method on a CEM Liberty microwave-
assisted automated synthesizer using Fmoc-Ala-Wang resin or
Fmoc-Val-Wang resin with an initial load of 0.4e0.8 mmol/g and
0.6 mmol/g (SigmaeAldrich), respectively. Standard Fmoc-chemistry
was used throughout.10 A solution of 20% piperidine in dime-
thylformamide (DMF) was used as deprotecting reagent, HBTU and
Scheme 7. Reaction of N-Fmoc-L-valyl-L-O-tert-butyl-serinyl-L-alanine Wang resin (18)
with dimethylsulfoxonium methylide (2).
DIPEA were used for coupling step. The peptide, N-Fmoc-
L-leucyl-L-
phenylalanyl- -alanine, was cleaved from the solid support with
L
trifluoroacetic acid (TFA) in the presence of triisopropylsilane (5%)
and water (5%) as scavengers. Reaction mixtures were monitored by
TLC, using silica gel 60-F254 percolated glass plates. The diethyl
ether solution of diazomethane was prepared from N-methyl-N-
nitrosourea following a classical procedure.11 The concentration of
the diazomethane solution (0.66 M) was obtained by a back titration
performed with a standard benzoic acid solution.12
short time and in good yields the amino function of N-Fmoc pro-
tected peptides and to perform simultaneously their cleavage from
Wang resin. Another experiment was designed to compare the ki-
netics of the deprotection reaction of the two functionalities, amino
and carboxyl group. To this aim, the N-Fmoc-L-alanine methyl ester
(1a) was treated with methylide 2 in a 1:1 molar ratio. The reaction
was monitored by TLC (chloroform/methanol 90:10 v/v). After only
5 min the formation of a product with a low Rf, that gave a positive
ninhydrin reaction, was observed. At the same time, an aliquot of
reaction mixture was hydrolyzed, extracted with an organic solvent
and derivatized with nosyl chloride. The crude reaction product was
analyzed by GC/MS. The GC/MS analysis revealed the presence of
4.2. Reaction of N-Fmoc-a-amino acid methyl esters (1aee)
with dimethylsulfoxonium methylide (2)
The appropriate N-Fmoc-a-amino acid methyl esters (1aee,
1 mmol) were added to a solution of the dimethylsulfoxonium
methylide (2, 2 mmol) in THF and the mixture was stirred at room
temperature under inert N2 atmosphere. The reaction monitored by
TLC (chloroform/methanol, 95:5 v/v) was completed after 20 min.
The mixture was evaporated to dryness under reduced pressure
and redissolved in 10 mL of water. The aqueous solution was
extracted with diethyl ether (3ꢀ10 mL). The corresponding
deprotected products 3aee were recovered as N-nosyl derivatives
only N-nosyl-
L
-alanine (4a) but did not show any peaks relative to
the N-nosyl- -alanine methyl ester. The product recovered, after only
L
5 min, was totally deprotected on both amino and carboxyl func-
tions. Therefore, we assumed that both deprotection reactions occur
with the same rate. After 30 min the reaction was stopped. The N-
Fmoc-
drolysis and extraction with organic solvent, while from acidic
aqueous solution was recovered the N-nosyl- -alanine in 55%. This
L-alanine methyl ester was recovered in 45% yield, after hy-
L
4aee. To this aim the aqueous solution containing the
a-amino
suggested that using 1 equiv of ylide with respect to the N-Fmoc
acids (3aee, 1 mmol) was allowed to react with nosyl chloride
(1 mmol) in dioxane at room temperature for 1 h. The mixture was
made basic with triethylamine (Et3N) (3 mmol). The mixture was
evaporated to dryness under reduced pressure. An aqueous solu-
tion of HCl 1 N was then added and the acidified solution (pH 2)
was extracted with ethyl acetate (3ꢀ10 mL). The organic layer was
dried (Na2SO4) and evaporated to dryness under reduced pressure
to give the corresponding N-nosyl derivatives 4aee.8a
protected methyl ester the reaction does not go to completion.
3. Conclusions
We have developed a new methodology to deprotect simulta-
neously the amino and carboxyl function of N-Fmoc-a-amino acid
and N-Fmoc-peptide methyl esters. The procedure involves the use of
dimethylsulfoxonium methylide and can be applied successfully to
both solution- and solid-phase peptide synthesis. In solid-phase
peptide synthesis the dimethylsulfoxonium methylide deprotects, in
short time and in good yields, the amino function of N-Fmoc pro-
tected peptides performing simultaneously theircleavage from Wang
resin. Both the deprotection reactions occur rapidly with the same
rate under mild conditions and do not cause any loss of the optical
integrityat the chiral centres of the peptide. Furthermore in solution-
phase peptide synthesis, the use of methylide as deprotecting reagent
of the amino function of N-Fmoc protected derivatives allows to
separate easily under basic conditions the dibenzofulvene (DBF), an
undesirable byproduct of the deprotection reaction. All these con-
siderations make this methodology a valid and important procedure
to obtain peptides free on both amino and carboxyl function at the
end of a synthetic process using the Fmoc-chemistry.
4.3. Synthesis of N-Fmoc-L-valine benzylamide (5)
Benzylamine (1.61 mmol) was added to a solution of the N-
Fmoc- -valine chloride (1.47 mmol) in dry dichloromethane
L
(15 mL) under N2. The resulting mixture was stirred at room tem-
perature for 1e2 h. The reaction mixture was then treated with an
aqueous solution of HCl 1 N. The organic phase was separated and
washed with a saturated solution of Na2CO3 (2ꢀ10 mL). The organic
extract was dried (Na2SO4) and evaporated to dryness to afford the
N-Fmoc-L-valine benzylamide (5).
4.3.1. N-Fmoc-
L
-valine benzylamide (5). Yield 90%. Found: C, 75.96;
H, 6.57; N, 6.56. C27H28N2O3 requires C, 75.68; H, 6.59; N, 6.54; Rf
(10% Et2O/CHCl3) 0.44; nmax (KBr) 3299, 3073, 2961, 1687, 1652,
1544,1452,1248,1033, 741, 695; dH (300 MHz, CDCl3) 0.86e0.98 (m,
6H, CH(CH3)2); 2.06e2.21 (m, 1H, CH(CH3)2); 3.98e4.08 (m, 1H,
4. Experimental section
4.1. General
PhCHaHbCH); 4.13e4.20 (m, 1H, PhCHaHbCH); 4.21e4.49 (m, 4H, a-
CH, CH2-Fmoc, CHeFmoc); 5.58 (d, 1H, J¼9.0 Hz, FmocNH);
6.55e6.62 (m, 1H, NH); 7.15e7.44 (m, 7H, C6H5CH2, ArHeFmoc),
7.35e7.45 (m, 2H, ArHeFmoc); 7.56 (d, 2H, J¼7.2 Hz, ArHeFmoc);
7.76 (d, 2H, J¼7.2 Hz, ArH); dC (75 MHz, CDCl3): 18.0, 19.3, 31.1, 43.5,
Solvents were purified and dried by standard procedures and
distilled prior to use. 1H NMR spectra were recorded at 300 MHz

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M. Spinella et al. / Tetrahedron 69 (2013) 2010e2016
47.1, 60.6, 67.0, 120.0, 125.0, 127.1, 127.4, 127.5, 127.7, 127.7, 128.7,
141.3, 143.8, 156.8, 171.0.
30 min. The basic conditions were maintained by a saturated
Na2CO3 aqueous solution, then the mixture was extracted with
chloroform (3ꢀ6 mL). The organic layer was washed with de-ion-
ized water (2ꢀ6 mL), then dried (Na2SO4) and evaporated to dry-
ness to afford the N-acetyl derivatives 10f and 10g in high yields.
4.4. Reaction of N-Fmoc-L-valine benzylamide (5) with
dimethylsulfoxonium methylide (2)
N-Fmoc-L-valine benzylamide 5 (1 mmol) was added to a solu-
4.6.1. N-Acetyl-L-alanyl-L-alanine (10f). Yield 90%. Found: C, 47.68;
tion of the dimethylsulfoxonium methylide (2, 2 mmol) in THF and
the mixture was stirred at room temperature under inert N2 at-
mosphere. The reaction monitored by TLC (chloroform/diethyl
ether, 90:10 v/v) was completed after 15 min. The mixture was
evaporated to dryness under reduced pressure and redissolved in
10 mL diethyl ether. The organic phase was treated with an aqueous
solution of HCl 1 N, the aqueous solution was basified with satu-
rated aqueous Na2CO3 and extracted with diethyl ether (3ꢀ10 mL).
The organic layer was dried with Na2SO4 and evaporated to dryness
H, 7.01; N, 13.83. C8H14N2O4 (202.21) requires C, 47.52; H, 6.98; N,
13.85.; Rf (10% CH3OH/CHCl3) 0.10; nmax (KBr) 3452e3100 (br), 2951,
2879, 1662, 1380, 1194, 959; dH (300 MHz, DMSO-d6) 1.16 (d, 3H,
J¼6.9 Hz, CHCH3); 1.26 (d, 3H, J¼6.9 Hz, CHCH3); 1.81 (s, 3H,
CH3CO); 4.13e4.20 (m, 1H, a-CH); 4.25e4.33 (m, 1H, a-CH); 8.09 (d,
1H, J¼7.5 Hz, NH); 8.20 (d, 1H, J¼6.9 Hz, NH); 12.50 (br s, 1H,
COOH); dC (75 MHz, DMSO-d6) 17.5, 18.7, 22.9, 47.8, 48.2, 171.7,
172.8, 174.4.
to give
L-valine benzylamide (7).
4.6.2. N-Acetyl-D-alanyl-L-alanine (10g). Yield 88%. Found: C, 47.67;
H, 6.96; N, 13.88. C8H14N2O4 requires C, 47.52; H, 6.98; N, 13.85; Rf
(10% CH3OH/CHCl3) 0.10; nmax (KBr) 3514e3000 (br), 2864, 1652,
1391, 1171, 986; dH (300 MHz, DMSO-d6) 1.16 (d, 3H, J¼6.9 Hz,
CHCH3); 1.24 (d, 3H, J¼6.9 Hz, CHCH3); 1.82 (s, 3H, CH3CO);
4.4.1.
L
-Valine benzylamide (7). Yield 90%. Found: C, 69.60; H, 8.83;
N, 13.62. C12H18N2O requires C, 69.87; H, 8.80; N, 13.58; Rf (10%
Et2O/CHCl3) 0.16; nmax (film) 3365, 3053, 2997, 1667, 1524, 1421,
1363, 741, 704; dH (300 MHz, CDCl3) 0.83 (d, 3H, J¼7.1 Hz,
CH(CH3)2); 0.99 (d, 3H, J¼7.1 Hz, CH(CH3)2); 1.66 (br s, 2H, NH2);
4.11e4.19 (m, 1H,
a-CH); 4.27e4.37 (m, 1H, a-CH); 8.01 (d, 1H,
J¼8.1 Hz, NH); 8.14 (d, 1H, J¼7.2 Hz, NH); dC (75 MHz, DMSO-d6):
2.28e2.39 (m, 1H, CH(CH3)2); 3.28 (d, 1H, J¼3.6 Hz,
a
-CH); 4.42
17.7, 19.0, 23.0, 47.8, 48.1, 143.8, 169.0, 172.3.
(dd,1H, J¼6.0, 15 Hz, PhCHaHbNH); 4.46 (dd,1H, J¼6.0 , 15 Hz,
PhCHaHbNH); 7.22e7.36 (m, 5H, C6H5CH2), 7.69 (br s, 1H, NH); dC
(75 MHz, CDCl3) 16.0, 19.7, 30.8, 43.1, 60.1, 127.3, 127.7, 128.6,
138.6, 174.2.
4.7. Synthesis of methyl ester of N-Fmoc-L-leucyl-L-phenyl-
alanyl- -alanine (11)
L
A 0.66 M solution of diazomethane2 in diethyl ether (8 mmol)
4.5. Synthesis of N-Fmoc-
L
-alanyl-
L
-alanine methyl ester and
was added cautiously dropwise to a stirred solution of the N-Fmoc-
N-Fmoc- -alanyl- -alanine methyl ester (8fand 8g)
D
L
L-leucyl-L-phenylalanyl-L-alanine (10, 1 mmol) synthesized by au-
tomated synthesizer in dry diethyl ether (10 mL). The resulting
mixture was maintained under an inert atmosphere (N2) and stir-
red at room temperature. TLC analysis (chloroform/methanol,
80:20 v/v) showed complete conversion of the precursor after
10 min. Evaporation of the solvent under reduced pressure afforded
To a magnetically stirred solution of the L-alanine methyl ester
hydrochloride (1 mmol) in 5% aqueous Na2CO3 (7 mL) was added
dropwise the appropriate N-Fmoc amino acid chloride (0.8 mmol)
in dry chloroform (10 mL).6a The resulting mixture was stirred for
2e3 h, monitoring the conversion of N-Fmoc-
L
-amino acid chloride
the N-Fmoc-L-leucyl-L-phenylalanyl-L-alanine methyl ester 11 in
by TLC (chloroform/methanol, 95:5). The organic phase was sepa-
rated and washed with 1 N HCl (2ꢀ5 mL). The organic extract was
washed with brine, then dried (Na2SO4) and evaporated to dryness
to give compounds 8f and 8g, in 84e92% overall yield.
90% yield.
4.7.1. N-Fmoc-
L
-leucyl-
L
-phenylalanyl-
L
-alanine
methyl
ester
(11). Yield 90%. Found: C, 70.02; H, 6.73; N, 7.20. C34H39N3O6 re-
quires C, 69.72; H, 6.71; N, 7.17. Rf (10% CH3OH/CHCl3) 0.90; nmax
(KBr) 3442, 3056, 2965, 1717, 1667, 1261, 1023, 744, 703; dH
(300 MHz, CDCl3) 0.81e0.91 (m, 6H, CH(CH3)2); 1.03e1.07 (m, 1H,
CH(CH3)2); 1.17e1.49 (m, 5H, CHCH3, CH2CH(CH3)2); 3.01e3.09 (m,
2H, CH2C6H5); 3.60 (s, 3H, OCH3); 4.12e4.25 (m, 2H, CHeFmoc,
CHleu); 4.25e4.35 (m, 1H, -CHphe); 4.40e4.55 (m, 2H, CH2eFmoc);
4.78e4.89 (m, 1H,
(d, 1H, J¼7.8 Hz, NH); 6.82 (d, 1H, J¼7.8 Hz, NH); 7.12e7.20 (m, 5H,
C6H5CH2); 7.25e7.36 (m, 2H, ArHeFmoc); 7.37e7.48 (m, 2H,
ArHeFmoc); 7.58 (d, 2H, J¼7.2 Hz, ArHeFmoc); 7.71e7.82 (m, 2H,
ArHeFmoc).
4.5.1. N-Fmoc-L-Alanyl-L-alanine methyl ester (8f). Yield 95%.
Found: C, 66.87; H, 6.08; N, 7.08. C22H24N2O5 requires C, 66.65; H,
6.10; N, 7.07; dH (300 MHz, CDCl3) 1.33e1.49 (m, 6H, CHCH3); 3.76
(s, 3H, OCH3); 4.17e4.36 (m, 2H, CH-Fmoc, CHCONH); 4.39 (d, 2H,
J¼6.9 Hz, CH2eFmoc); 4.52e4.62 (m, 1H,CHCOOMe); 5.51 (br s, 1H,
NHeFmoc); 6.62 (br s, 1H, CONH); 7.29e7.43 (m, 4H, ArH);
7.58e7.64 (m, 2H, ArH); 7.77 (d, 2H, J¼7.5 Hz, ArH); dC (75 MHz,
CDCl3) 18.3, 18.8, 47.1, 48.1, 50.4, 52.6, 67.1, 120.0, 125.1, 127.1, 127.6,
141.3, 143.7, 155.9, 171.8, 173.1.
a-
a
a
-CHala); 5.30 (d, 1H, J¼7.8 Hz, NHeFmoc); 6.66
4.6. Reaction of N-Fmoc-dipeptides methyl esters 8fand 8g
with dimethylsulfoxonium methylide (2)
4.8. Reaction of N-Fmoc-L-leucyl-L-phenylalanyl-L-alanine
methyl ester (11) with dimethylsulfoxonium methylide (2)
The appropriate N-Fmoc-dipeptide methyl esters 8f and 8g
(1 mmol) were added to a solution of the dimethylsulfoxonium
methylide (2, 2 mmol) in THF and the mixture was stirred at room
temperature under inert N2 atmosphere. The reaction monitored by
TLC (chloroform/methanol, 90:10 v/v) was completed after 20 min.
The mixture was evaporated to dryness under reduced pressure
and redissolved in 10 mL of water. The aqueous solution was
extracted with diethyl ether (3ꢀ10 mL). The products 9f and 9g
were recovered as N-Acetyl derivatives. The aqueous solution
containing the dipeptides 8f and 8g was treated with acetic anhy-
dride (6 mmol) in chloroform (10 mL) at room temperature for
The N-Fmoc-tripeptide methyl ester 11 (1 mmol) was added to
a solution of the dimethylsulfoxonium methylide (2, 2 mmol) in
THF and the mixture was stirred at room temperature under inert
N2 atmosphere. The reaction monitored by TLC (chloroform/
methanol 90:10 v/v) was completed after 20 min. The mixture was
evaporated to dryness under reduced pressure and redissolved in
10 mL of water. The aqueous solution was extracted with diethyl
ether (3ꢀ10 mL). The deprotected product 12 was recovered as N-
nosyl derivative. The aqueous solution containing the tripeptide 12
(1 mmol) was allowed to react with nosyl chloride (1 mmol) in

M. Spinella et al. / Tetrahedron 69 (2013) 2010e2016
2015
dioxane at room temperature for 1 h. The mixture was made basic
with triethylamine (Et3N) (3 mmol). The mixture was evaporated to
dryness under reduced pressure. An aqueous solution of HCl 1 N
was then added and the acidified solution (pH 2) was extracted
with ethyl acetate (3ꢀ10 mL). The organic layer was dried (Na2SO4)
and evaporated to dryness under reduced pressure to give N-nosyl
derivative 13.
4.11.1. N-Acetyl-
L
-alanyl-
L
-valine (17). Yield 90%. Found: C, 52.08;
H, 7.91; N, 12.20. C10H18N2O4 requires C, 52.16; H, 7.88; N, 12.17; Rf
(10% CH3OH/CHCl3) 0.12; nmax (KBr) 3504e3000 (br), 1662, 1237,
1013, 823; dH (300 MHz, DMSO-d6) 0.86 (d, 6H, J¼6.8 Hz,
CH(CH3)2); 1.17 (d, 3H, J¼6.8 Hz, CHCH3); 1.91 (s, 3H, CH3CONH);
1.98e2.09 (m, 1H, CH(CH3)2); 3.97e4.20 (m, 1H,
a-CHval);
4.30e4.41 (m, 1H,
J¼10.6 Hz, NH).
a-CHala), 7.86 (d, 1H, J¼10.6 Hz, NH), 8.11 (d, 1H,
4.8.1. N-Nosyl-L-leucyl-L-phenylalanyl-L-alanine (13). Yield 80%.
Found: C, 54.13; H, 5.68; N, 10.44. C24H30N4O8S requires C, 53.92; H,
5.66; N, 10.48; Rf (10% CH3OH/CHCl3) 0.22; nmax (KBr) 3432e3100
(br), 3063, 2961, 1662, 1299, 1156, 823, 751; dH (300 MHz, CDCl3)
0.61 (d, 3H, J¼6.5 Hz, CH(CH3)2); 0.79 (d, 3H, J¼6.5 Hz, CH(CH3)2;
0.81e0.98 (m, 2H, CH2CH(CH3)2); 1.23e1.49 (m, 4H, CH(CH3)2,
CHCH3); 2.90e3.01 (m, 1H, CHaHbC6H5); 3.10e3.21 (m, 1H,
4.12. Reaction of N-Fmoc-
nine Wang resin (18) with dimethylsulfoxonium methylide (2)
L-valyl-O-tert-butyl-L-serinyl-L-ala-
N-Fmoc-L-valyl-O-tert-butyl-L-serinyl-L-alanine Wang resin (18,
1 mmol) was added to the solution of the dimethylsulfoxonium
methylide (2, 2 mmol) in THF and the mixture was stirred at room
temperature under inert N2 atmosphere. The reaction monitored by
TLC (chloroform/methanol, 90:10 v/v) was completed after 20 min.
The mixture was hydrolyzed and filtered. After evaporation of THF
under reduced pressure, the filtrate was extracted with diethyl
ether (3ꢀ10 mL). The aqueous solution containing the tripeptide
CHaHbC6H5); 3.69e3.84 (m, 1H,
a-CHleu); 4.38e4.49 (m, 1H, a-
CHala); 4.67e4.78 (m, 1H, -CHphe); 6.31 (br s, 1H, NH); 7.11 (d, 1H,
a
J¼9.0 Hz, NH); 7.15e7.38 (m, 5H, C6H5CH2); 7.44 (d, 1H, J¼9.0 Hz,
NH); 8.05 (d, 2H, J¼9.3 Hz, ArHeNs); 8.28 (d, 2H, J¼9.3 Hz,
ArHeNs). dC (75 MHz, CDCl3): 17.5, 21.4, 23.3, 24.2, 37.8, 40.8, 47.9,
53.9, 55.5, 124.4, 126.7, 128.4, 129.8, 138.1, 146.9, 149.5, 170.9,
171.0, 174.3.
L-valyl-O-tert-butyl-L-serinyl-L-alanine (19) was allowed to react
with nosyl chloride (1 mmol) in dioxane at room temperature
for 1 h. The mixture was made basic with triethylamine (Et3N)
(3 mmol). The mixture was then evaporated to dryness under re-
duced pressure, acidified with a 5% NaHSO4 aqueous solution (pH
5) and extracted with ethyl acetate (3ꢀ10 mL). The organic layer
was dried (Na2SO4) and evaporated to dryness under reduced
4.9. Synthesis of N-nosyl-L-Ala Wang resin (14)
The Wang resin (1.1 mmol OH/g; 100e150 mg) was swelled by
stirring at 25 ꢁC for 10 min in 2e3 mL of dichloromethane. The
solvent was removed by filtration. The activated resin was treated
pressure to give pure N-acetyl-
nine 20 in 80% yield.
L-valyl-O-tert-butyl-L-serinyl-L-ala-
with a solution of N-nosyl-L-alanine chloride in dry dichloro-
methane (3 mL) and pyridine (6 mmol). The mixture was stirred for
3e5 h. The resin was then filtered and washed with dichloro-
methane (3ꢀ5 mL).
4.12.1. N-Nosyl-L-valyl-O-tert-butyl-L-serinyl-L-alanine (20). Yield
80%. Found: C, 49.02; H, 6.26; N, 10.82. C21H32N4O9S requires C,
48.83; H, 6.24; N, 10.85; Rf (10% CH3OH/CHCl3) 0.16; nmax (KBr)
3467e3000 (br), 2864, 1657, 1530, 1349, 1222, 1043, 997, 833, 772;
dH (300 MHz, DMSO-d6) 0.82 (d, 6H, J¼7.0 Hz, CH(CH3)2); 1.22 (d,
3H, J¼7.2 Hz, CHCH3); 1.32 (s, 9H, (CH3)3C); 1.75e1.98 (m, 1H,
CH(CH3)2); 3.34 (d, 2H, J¼6.3 Hz, CH2O-t-Bu); 3.70 (dd, 1H, J¼7.2,
4.10. Reaction of N-nosyl-L-Ala Wang resin (14) with dime-
thylsulfoxonium methylide (2)
The N-nosyl- -Ala Wang resin (14, 1 mmol) was added to a so-
L
lution of the dimethylsulfoxonium methylide in THF (2, 2 mmol)
and the mixture was stirred at room temperature under inert N2
atmosphere. The reaction monitored by TLC (chloroform/methanol,
90:10 v/v) was completed after 20 min. The mixture reaction was
filtered and washed with THF. The filtrate was evaporated to dry-
ness under reduced pressure. The filtrate was basified with NaOH
1 N and extracted with diethyl ether (3ꢀ10 mL). The aqueous so-
lution was acidified with HCl 1 N and extracted with diethyl ether.
9.3 Hz, a-CHval); 3.89e4.10 (m, 1H, a-CHser); 4.11e4.25 (m, 1H, a-
CHala); 7.92 (d, 1H, J¼7.2 Hz, NH); 8.01 (d, 2H, J¼8.4 Hz, ArH); 8.04
(d, 1H, J¼7.8 Hz, NH); 8.23 (d, 1H, J¼9.3 Hz, NH); 8.34 (d, 2H,
J¼8.4 Hz, ArH); 12.50 (br s, 1H, COOH); dC (75 MHz, DMSO-d6): 17.7,
18.7, 19.4, 29.0, 31.4, 48.0, 55.1, 61.9, 62.3, 124.5, 128.6, 147.2, 149.7,
169.7, 170.1, 174.4.
The solvent was evaporated to afford the N-nosyl-L-Ala-OH (4a) as
4.13. Reaction of N-Fmoc-L-alanine methyl ester (1a) with
yellow solid in 80% overall yield.
dimethylsulfoxonium methylide (2) in 1:1 molar ratio
4.11. Reaction of N-Fmoc-
L
-alanyl-
L
-valine Wang resin (15)
N-Fmoc-L-alanine methyl ester (1a, 1 mmol) was added to
with dimethylsulfoxonium methylide (2)
a solution of the dimethylsulfoxonium methylide (2, 1 mmol) and
the mixture was stirred at room temperature under inert N2 at-
mosphere in THF. After 30 min the reaction, analyzed by TLC
(chloroform/methanol, 95:5 v/v), does not go to completion.
However, the mixture was evaporated to dryness under reduced
pressure, redissolved in 10 mL of water and extracted with diethyl
ether. The organic layer, dried (Na2SO4), was evaporated to dry-
N-Fmoc-L-alanyl-L-valine Wang resin (15) (1 mmol) was added
to the solution of the dimethylsulfoxonium methylide in THF (2,
2 mmol) and the mixture was stirred at room temperature under
inert N2 atmosphere. The reaction monitored by TLC (chloroform/
methanol, 90:10 v/v) was completed after 20 min. The mixture was
hydrolyzed and filtered. After evaporation of THF under reduced
pressure, the filtrate was extracted with diethyl ether (3ꢀ10 mL).
The aqueous solution containing the deprotected dipeptide 16 was
treated with acetic anhydride (6 mmol) in chloroform (10 mL) at
room temperature for 30 min. The basic conditions were main-
tained by adding of a saturated NaHCO3 aqueous solution. The
mixture was acidified with HCl 1 N and extracted with chloroform
(3ꢀ6 mL). The organic layer was washed with distilled water
(2ꢀ6 mL), then dried (Na2SO4) and evaporated to dryness to afford
ness under reduced pressure to give the N-Fmoc-L-alanine methyl
ester in 45% yield. The aqueous solution was derivatized with
nosyl chloride (1 mmol) and triethylamine (Et3N) (3 mmol) in
dioxane at room temperature for 1 h. The mixture after evapo-
ration to dryness under reduced pressure was acidified with
aqueous solution of HCl 1 N (pH 2) and extracted with ethyl ac-
etate (3ꢀ10 mL). The organic layer was dried (Na2SO4) and
evaporated to dryness under reduced pressure to give the corre-
sponding N-nosyl-
L
-alanine (4a) in 55% yield. The product 4a was
N-acetyl-L-alanyl-L-valine (17) in 90% yield.
analyzed by GC/MS.

2016
M. Spinella et al. / Tetrahedron 69 (2013) 2010e2016
3. (a) Kociensky, P. J. Protecting Groups, 3rd ed.; Georg Thieme: Germany, 2005; (b)
4.13.1. N-Nosyl-
-alanine (4a). GCeMS (E.I.) m/z: 274 (Mꢂþ, 1%), 229
(100), 186 (33), 122 (19).
L
Pearson, A. J.; Roush, W. R. Handbook of Reagents for Organic Synthesis, Acti-
vating Agents and Protecting Groups; Wiley: Chichester, UK, 1999.
4. (a) Bayermann, M.; Biernet, M.; Niedrich, H.; Carpino, L. A.; Sadat-Aalaee, D. J.
Org. Chem. 1990, 55, 721; (b) Carpino, L. A.; Sadat-Aalaee, D.; Bayermann, M. J.
Org. Chem. 1990, 55, 1673.
Supplementary data
5. Carpino, L. A.; Cohen, B. J.; Stephens, K. E., Jr.; Sadat-Aalaee, S. Y.; Tien, J. H.;
Langridge, D. C. J. Org. Chem. 1986, 51, 3734.
6. (a) Leggio, A.; Liguori, A.; Napoli, A.; Siciliano, C.; Sindona, G. Eur. J. Org. Chem.
2000, 573; (b) Di Gioia, M. L.; Leggio, A.; Le Pera, A.; Liguori, A.; Perri, F.;
Siciliano, C. Eur. J. Org. Chem. 2004, 4437; (c) Di Gioia, M. L.; Leggio, A.; Le Pera,
A.; Siciliano, C.; Liguori, A. J. Pept. Res. 2004, 63, 383.
Copies of the 1H NMR and 13C NMR spectra of compounds 4aee,
5, 7, 8f, 10f,g, 13, 20. Copies of the 1H NMR spectra of compounds 11
and 17. Supplementary data associated with this article can be
found in the online version, at http://dx.doi.org/10.1016/
j.tet.2012.12.052.
7. Leggio, A.; De Marco, R.; Perri, F.; Spinella, M.; Liguori, A. Eur. J. Org. Chem.
2012, 114.
8. (a) Di Gioia, M. L.; Leggio, A.; Liguori, A. J. Org. Chem. 2005, 70, 3892; (b) Leggio,
A.; Di Gioia, M. L.; Perri, F.; Liguori, A. Tetrahedron 2007, 63, 8164.
9. Meshram, H. M.; Reddy, G. S.; Reddy, M. M.; Yadav, J. S. Tetrahedron Lett. 1998,
39, 4103.
References and notes
10. Fields, G. B.; Noble, R. L. Int. J. Peptide Protein Res. 1990, 35, 161.
11. Leggio, A.; Liguori, A.; Napoli, A.; Siciliano, C.; Sindona, G. J. Org. Chem. 2001,
66, 2246.
1. Wuts, P. G. M.; Greene, T. W. Greene’s Protective Groups in Organic Synthesis, 4rd
ed.; Wiley: New York, NY, 2007.
2. Nicolaou, K. C. R.; Groneberg, D.; Miyazaki, T.; Stylianides, N. A. T.; Schulze, J.;
Stahl, W. J. Am. Chem. Soc. 1990, 112, 8193.
12. Arndt, F. Org. Synth. 1934, 2, 165.