α-N-Protected dipeptide acids: A simple and efficient synthesis via the easily accessible mixed anhydride method using free amino acids in DMSO and tetrabutylammonium hydroxide

Article abstract of DOI:10.1002/psc.2503

The importance of dipeptides both in medicinal and pharmacological fields is well documented and many efforts have been made to find simple and efficient methods for their synthesis. For this reason, we have investigated the synthesis of α-N-protected dipeptide acids by reacting the easily accessible mixed anhydride of α-N-protected amino acids with free amino acids under different reaction conditions. The combination of TBA-OH and DMSO has been found to be the best to overcome the low solubility of amino acids in organic solvents. Under these experimental conditions, the homogeneous phase condensation reaction occurs rapidly and without detectable epimerization. The present method is also applicable to side-chain unprotected Tyr, Trp, Glu, and Asp but not Lys. This latter residue is able to engage two molecules of mixed anhydride giving the corresponding isotripeptide. Moreover, the applicability of this protocol for the synthesis of tri- and tetrapeptides has been tested. This approach reduces the need for protecting groups, is cost effective, scalable, and yields dipeptide acids that can be used as building blocks in the synthesis of larger peptides.

Full text of DOI:10.1002/psc.2503

Research Article
Received: 2 January 2012
Revised: 2 January 2013
Accepted: 2 January 2013
Published online in Wiley Online Library: 14 March 2013
(wileyonlinelibrary.com) DOI 10.1002/psc.2503
a-N-Protected dipeptide acids: a simple
and efficient synthesis via the easily
accessible mixed anhydride method
using free amino acids in DMSO and
tetrabutylammonium hydroxide
G. Verardoa* and A. Gorassinib
The importance of dipeptides both in medicinal and pharmacological fields is well documented and many efforts have
been made to find simple and efficient methods for their synthesis. For this reason, we have investigated the synthesis of
a-N-protected dipeptide acids by reacting the easily accessible mixed anhydride of a-N-protected amino acids with free amino
acids under different reaction conditions. The combination of TBA-OH and DMSO has been found to be the best to overcome
the low solubility of amino acids in organic solvents. Under these experimental conditions, the homogeneous phase conden-
sation reaction occurs rapidly and without detectable epimerization. The present method is also applicable to side-chain
unprotected Tyr, Trp, Glu, and Asp but not Lys. This latter residue is able to engage two molecules of mixed anhydride
giving the corresponding isotripeptide. Moreover, the applicability of this protocol for the synthesis of tri- and
tetrapeptides has been tested. This approach reduces the need for protecting groups, is cost effective, scalable, and yields
dipeptide acids that can be used as building blocks in the synthesis of larger peptides. Copyright © 2013 European Peptide
Society and John Wiley & Sons, Ltd.
Characterization data of all compounds synthesized are given in the Supporting Information.
Keywords: a-N-protected dipeptide acids; mixed carbonic carboxylic anhydride; free amino acids; tetrabutylammonium hydroxide;
epimerization; isotripeptides
2,3,4,5,6-pentafluorophenol [49–51], 1-hydroxybenzotriazole [52–56],
benzisoxazolium salts [57,58], N-hydroxysuccinimide [59,60],
Introduction
Dipeptides and small peptides have wide applications both in
medicinal and synthetic chemistry. For example, they are enzyme
inhibitors [1–3], prodrugs [4–12], peroxisome proliferator-activated
receptor gamma antagonist [13], anti-HIV-1 [14], and chemothera-
peutic metallopharmaceuticals [15–21]. Dipeptides are also used
as additional supplementation of single important amino
acid nutrients like glutamine [22–29], tyrosine [25,30,31], and
cysteine [25]. Moreover, they provide one-dimensional nanostruc-
tured materials [32–35], ecofriendly anionic surfactants [36], and
catalysts for the direct asymmetric aldol reaction [37–40].
In the solution phase synthesis of a peptide bond, the a-amino
and the a-carboxyl groups of the N-terminal and C-terminal,
respectively, amino acid residues must be protected before
activating the carboxyl group. The elongation of the peptide
chain involves the selective deprotection of the functional group
that is to enter into formation of the subsequent peptide bond
[41–45]. All these sequential steps are time-consuming, reduce
the overall yield, and sometimes may cause a small degree of
epimerization [46,47]. The possibility of using free amino acids
as the nucleophile may reduce some of these drawbacks.
and N-hydroxy-3-azaspiro[5,5]undecane-2,4-dione [61] have been
employed to this purpose. These intermediates are stable and
the coupling reaction can be carried out under the experimental
conditions suitable to dissolve the free amino acids (water with or
without an organic cosolvent, presence of an inorganic or organic
base). To avoid the use of water, some authors proposed a system
consisting of a strong acid [62] or a neutral salt [63,64] in the
presence of a tertiary amine to increase the solubility of free amino
acids in DMF.
Always with the aim to increase the solubility of amino acids in
organic solvents (MeCN, DMF, CHCl3, EtOAc), methods employing
phosphazene bases (Schwesinger bases) [65], phase-transfer
reagents [66–68], alkaline earth metal ions [69], and ultrasound
in the presence of TEA [70] have also been reported. These
*
Correspondence to: Giancarlo Verardo, Dipartimento di Chimica, Fisica
e Ambiente, Università di Udine, Via del Cotonificio 108, I-33100 Udine, Italy.
E-mail: giancarlo.verardo@uniud.it
a Dipartimento di Chimica, Fisica e Ambiente, Università di Udine, Via del
Cotonificio 108, I-33100 Udine, Italy
There are many reports on the coupling reaction between free
and a-N-protected amino acids with different a-carboxyl activat-
ing agents. The active ester has been by far the most used: both
phenols and N-hydroxy compounds such as 4-nitro- [13,48],
b Dipartimento di Storia e Tutela dei Beni Culturali, Università di Udine, Palazzo
Caiselli, Vicolo Florio 2, I-33100 Udine, Italy
J. Pept. Sci. 2013; 19: 315–324
Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

VERARDO AND GORASSINI
experimental protocols allow to carry out the coupling reaction
of free amino acids with N-carboxyamino acid anhydrides [70]
or with a-N-protected amino acids activated as esters [65,67–70]
or acyl azides [66,68] in a water-free system. Recently, Brown
et al. [71] reported the synthesis of some Fmoc-dipeptides
combining Fmoc-amino acid fluorides with an excess of unpro-
tected amino acids dissolved in hexafluoropropanol.
a mixture of Z-Phe-OH (1Zf, 1.0mmol), ethyl chloroformate
(1.4mmol), and TEA (3.0mmol) in THF (20 ml) was stirred for
30 min at 0 ꢀC. After this time, a solution of H-Phe-OH (3f, 1.5 mmol)
in H2O (20 ml) was added to the resulted suspension and the stir-
ring was continued for 30 min at 0ꢀC. After suitable work-up [72],
the crude product (0.457 g) was crystallized [72] to afford only
1
0.281 g (63% yield) of 4Zff. H NMR analysis of the concentrated
In all the reported methods, the N-protected-carboxyl-
activated amino acid must be isolated and purified from the
reaction by-products and some require expensive and/or toxic
reagents (e.g., phosphazene bases, diethylaminosulfur trifluoride,
and triphosgene). Moreover, the use of phase-transfer reagents
or alkaline earth metal ions require the lyophilization of the
amino acids salts obtained thus resulting in more expensive
and time-consuming processes.
It is noteworthy that the easily accessible mixed carbonic
carboxylic anhydride method has received little attention in
recent years. In fact, the few entries found in literature report
the reaction of basic aqueous solutions of amino acids only with
N-protected proline [17,37] (low yields) and, more recently, with
N-protected phenylalanine [72]. The last one seemed the most
appealing for our purposes, but we have encountered some
problems (shown in later text) to reproduce the yields reported
for some of the reactions described and to extend the protocol
to other N-protected amino acids. All these reasons prompted
us to develop a more versatile approach to the synthesis of
N-protected dipeptide acids using the mixed anhydride method
to activate the carboxyl group of N-protected amino acids.
crystallization mother liquor (0.175 g) evidenced the presence of
Z-Phe-OH (1Zf), N-ethoxycarbonylphenylalanine, and 4Zff in 63%,
32%, and 5% yields, respectively. The large amount of water,
the presence of TEA, and the excess of ethyl chloroformate used
during the reaction were responsible for the formation of 1Zf and
N-ethoxycarbonylphenylalanine. Similar or worse results were
obtained for the synthesis of Fmoc-Phe-Val-OH (4Ffv), Boc-
Leu-Phe-OH (4Blf), and Fmoc-Leu-Ala-OH (4Fla). On the basis
of these results, we have investigated, with the aid of ESI–MS, the
reaction of H-Phe-OH (3f) with the mixed anhydride 2Zf obtained
from Z-Phe-OH (1Zf, 1.0 mmol) and isobutyl chloroformate (IBCF,
1.03 mmol) prepared in THF (10 ml) in the presence of NMM
(1.03 mmol) at 10 ꢀC (Table 1). First of all, we reduced the amount
of water to dissolve 3f (1.2mmol) using 1.6 M NaOH (1.2mmol).
The addition of this solution to the mixed anhydride 2Zf caused
the partial precipitation of sodium phenylalaninate, and the ESI–
MS analysis of the crude mixture after 30 min at 0 ꢀC showed the
presence of the tripeptide Z-Phe-Phe-Phe-OH (5Zfff, 42%), the
starting Z-Phe-OH (1Zf, 20%), and the desired dipeptide 4Zff which
was isolated in 15% yield (Table 1, entry 1). The low concentration
of phenylalaninate in THF, because of its partial precipitation and
the presence of the strong base NaOH, allowed the displacement
of the isobutyloxycarbonyl group from the unreacted 2Zf to the
sodium salt of dipeptide 4Zff giving the corresponding mixed
anhydride 6Zff which competed with 2Zf for 3f producing the
tripeptide 5Zfff (Scheme 1). In fact, when tetrabutylammonium
Results and Discussion
The first approach to this study was the synthesis of Z-Phe-Phe-OH
(4Zff) according to the method proposed by Noguchi et al. [72]:
Table 1. Experimental conditions optimization for the synthesis of a-N-protected dipeptide acids 4 by the reaction of mixed anhydride 2Zf and
free amino acids 3
Entry
Solventb (ml)
Base (mmol)
3 (mmol)
Additive (mmol)
Reaction
Yieldc of
Yieldc of 5 (%)
Yieldd of 4 (%)
time (min)
1Zf (%)
1
2
3
4
5
6
H2O (0.75)
H2O (0.75)
H2O (5.24)
H2O (5.24)
H2O (4.38)
DMSO (3.50)
H2O (0.52)
DMSO (3.50)
H2O (0.52)
NaOH (1.2)
NaOH (1.2)
NaOH (1.1)
NaOH (1.1)
TBA–OH (1.1)
TBA–OH (1.1)
3f (1.2)
3f (1.2)
3f (1.1)
3a (1.1)
3a (1.1)
3f (1.2)
30
30
20
10
8
5Zfff
42
20
4
4Zff 15
4Zff 42
4Zff 73
4Zfa 64
4Zfa 74
4Zff 82
TBA-Br (1.2)
KH2PO4 (5.4)
KH2PO4 (5.4)
KH2PO4 (5.4)
5Zfff
5Zfff
5Zfaa
5Zfaa
5Zfff
120
120
60
14
7
5
4
30
3
4
7
TBA–OH (1.1)
3f (1.2)
30
3
5Zfff
no detect.
4Zff 91
aThe synthesis of the mixed anhydride 2Zf was carried out by adding IBCF (1.03 mmol) to a THF (10 ml) solution of 1Zf (1.0 mmol) and NMM
(1.03 mmol) at ꢁ15 ꢀC. The reaction mixture was stirred for 10 min at ꢁ15 ꢀC before adding 3.
bType and amount of solvent used to dissolve 3.
cDetermined by ESI–MS analysis.
dIsolated yield.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2013; 19: 315–324

SYNTHESIS OF a-N-PROTECTED DIPEPTIDE ACIDS
O
O
O
Z
COONa
R
Ph
O
O
Ph
N
H
+
N
N
H
Z
H
2Zf
4Zfa (sodium salt): R = CH3
4Zff (sodium salt): R = PhCH2
H
N
O
O
R
O
O
R
COONa
O
Z
O
Z
+ 3a or 3f
Ph
+
O
COONa
N
Ph
N
H
Ph
N
H
R
H
Z
N
N
H
1Zf (sodium salt)
H
6Zfa: R = CH3
6Zff: R = PhCH2
5Zfaa (sodium salt): R = CH3
5Zfff (sodium salt): R = PhCH2
Scheme 1. Formation of tripeptides 5Zfaa and 5Zfff.
bromide (TBA-Br, 1.2mmol) was added to increase the solubility of
phenylalaninate in THF, the yields of tripeptide 5Zfff and Z-
Phe-OH (1Zf) decreased to 20% and 10%, respectively, and
Z-Phe-Phe-OH (4Zff) was isolated in 42% yield (Table 1, entry
2). However, also in this case, the presence of NaOH could
cause the formation of Z–Phe–Phe–Phe–OH (5Zfff) promoting
the route described in Scheme 1. To verify this hypothesis,
the coupling reaction was carried out at pH 6.5 by adding a
mixture of H-Phe-OH (3f, 1.1 mmol), 0.8 M NaOH (1.1 mmol),
and 1.4 M KH2PO4 (5.4 mmol) to the preformed mixed anhy-
dride 2Zf. The ESI–MS analysis of the intact reaction mixture
showed longer reaction times for the complete disappearance
of 2Zf (2 h), but the amount of Z-Phe-Phe-Phe-OH (5Zfff) was
reduced to 4% (Table 1, entry 3) and Z-Phe-Phe-OH (4Zff) was
isolated in 73% yield. Under these experimental conditions,
the substitution of H-Phe-OH (3f) with the more water soluble
H-Ala-OH (3a) caused an increase in the hydrolysis product
Z-Phe-OH (1Zf, 14% yield) affording the corresponding dipeptide
Z-Phe-Ala-OH (4Zfa) in 64% yield (Table 1, entry 4). To overcome
the low solubility of 3a in THF at pH 6.5, NaOH was substituted with
55% aqueous TBA–OH (1.1 mmol). In this case (Table 1, entry 5), the
coupling reaction of 3a with the preformed mixed anhydride 2Zf
was faster (1h), the extent of hydrolysis was reduced (7% yield),
and the dipeptide 4Zfa was isolated in 74% yield. Although this
method (Method A) provided better yields compared with those
obtained with the protocol of Noguchi et al. [72], some tripeptide
Z-Phe-Ala-Ala-OH (5Zfaa) was still present (Table 1, entry 5), and
the reaction with the high water-soluble aspartic (3d) and glutamic
acid (3e) gave the corresponding dipeptide 4 in low yield (Table 3).
The observation that DMSO was able to dissolve a mixture of
H-Phe-OH (3f, 1.2 mmol) and 55% aqueous TBA–OH
(1.1 mmol) allowed to carry out the reaction in the presence
of a large amount of 3f in the organic phase (THF–DMSO)
favoring the coupling mechanism leading to 4Zff (reaction time
30 min, 82% yield) over the hydrolysis of the mixed anhydride 2Zf
(3% yield; Table 1, entry 6). Finally (Table 1, entry 7), the removal
by filtration of the N-methylmorpholinium chloride formed in the
activation step gave a crude product with no detectable amount
of tripeptide 5Zfff (ESI–MS) and 91% of isolated yield of the desired
dipeptide 4Zff (Method B). On the basis of these experimental
findings, Method B was extended to the synthesis of other
dipeptides 4 (Table 2).
The obtained results as well as some properties of dipeptides
4 prepared are reported in Table 3. Some dipeptides 4 were
also synthesized according to Method A and the yields were
compared with those obtained from Method B (Table 3).
With the aid of ESI–MS analysis, we observed that the
reaction rate (30 min) was not influenced by the nature of both
the mixed anhydride 2 and the a-amino acid 3 involved.
However, when the reaction was carried out with a-N-protected
amino acids such as valine (1Bv, 1Fv), 2-methylalanine (1Bu,
1Zu), and proline (1Zp), the N-isobutyloxycarbonyl 7 of the
amino acid 3 was present in 7%–9% yield. The absence of
unreacted N-protected amino acid 1 in the corresponding
mixed anhydrides 2Bv, 2Fv, 2Bu, 2Zu, and 2Zp highlighted
the complete consumption of IBCF during the synthesis of 2.
Therefore, both the steric hindrance of the amino acid side
chain (Val, Aib, Pro) and the high reactivity of the TBA salt of
3 (3-TBA) resulted in the formation of isobutylcarbamate 7 via
the reaction of 3-TBA with the carboxyl carbon of the carbonic
acid ester moiety of the mixed anhydride (Scheme 2) [74,75]. In
fact, the substitution of IBCF with the more sterically hindered
isopropyl chloroformate [76] produced less isopropylcarbamate
8 (3%–4%) and increased the yield of the corresponding
dipeptide 4 (Scheme 2; Table 3).
The amount of TBA–OH employed was critical because even a
slight excess produced the N-isobutyloxycarbonyl 9 [77] of the
starting N-protected amino acid 1 via the corresponding mixed
anhydride 2 (Scheme 3). For this reason, less than one equivalent
of TBA–OH with respect to 3 was used even with amino acids
bearing acidic functional groups such as tyrosine (3y), aspartic
(3d), and glutamic (3e) acid. When the reaction between the
mixed anhydride of N-benzyloxycarbonylphenylalanine (2Zf;
1.0 equiv) and tyrosine (3y; 1.2 equiv) was carried out in the presence
of an excess of TBA–OH (2.4 equiv), the ESI–MS analysis of the intact
reaction mixture showed, in addition to the dipeptide Z-Phe-Tyr-OH
(4Zfy; 55% yield) and the expected N-isobutyloxycarbonyl-
N-benzyloxycarbonylphenylalanine (9Zf; 10% yield), the presence
of N,O-bis-(carbobenzyloxyphenylalanyl)-tyrosine (10; 35% yield)
because of the reaction of two molecules of 2Zf with both the
a-amino and 4-hydroxyphenol groups of tyrosine (3y). The yield
of 10 raised to 72% (isolated yield 32%) when the reaction was
performed using 3y, TBA–OH, and 2Zf in the molar ratio of
0.5 : 1.0: 1.0, respectively.
J. Pept. Sci. 2013; 19: 315–324 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

VERARDO AND GORASSINI
Table 2. Synthesis of a-N-protected dipeptide acids 4
1, 2
Bf
Bf
Bf
Bf
Ba
Bl
PG-AA1-
Boc-Phe-
Boc-Phe-
Boc-Phe-
Boc-Phe-
Boc-Ala-
Boc-Leu-
Boc-Leu-
Boc-Aib-
Boc-Val-
Boc-Val-
Eoc-Phg-
Fmoc-Phe-
Fmoc-Phe-
Fmoc-Phe-
Fmoc-Phe-
Fmoc-Phe-
Fmoc-Leu-
Fmoc-Leu-
Fmoc-Leu-
Fmoc-Val-
Fmoc-Met-
Fmoc-Trp-
Z-Phe-
3
a
y
d
e
y
f
H-AA2-OH
H-Ala-OH
H-Tyr-OH
H-Asp-OH
H-Glu-OH
H-Tyr-OH
H-Phe-OH
H-Gln-OH
H-Phe-OH
H-Phe-OH
H-Trp-OH
H-Ala-OH
H-Ala-OH
H-Phe-OH
H-Leu-OH
H-Val-OH
H-Phg-OH
H-Ala-OH
H-Phg-OH
H-Aib-OH
H-Asp-OH
H-Glu-OH
H-Thr-OH
H-Ala-OH
H-Tyr-OH
H-Phe-OH
H-Gln-OH
H-Leu-OH
H-Aib-OH
H-Tyr-OH
H-Glu-OH
H-Phe-OH
H-Phg-OH
H-Phe-OH
4
PG-AA1-AA2-OH
Boc-Phe-Ala-OH
Boc-Phe-Tyr-OH
Boc-Phe-Asp-OH
Boc-Phe-Glu-OH
Boc-Ala-Tyr-OH
Boc-Leu-Phe-OH
Boc-Leu-Gln-OH
Boc-Aib-Phe-OH
Boc-Val-Phe-OH
Boc-Val-Trp-OH
Eoc-Phg-Ala-OH
Fmoc-Phe-Ala-OH
Fmoc-Phe-Phe-OH
Fmoc-Phe-Leu-OH
Fmoc-Phe-Val-OH
Fmoc-Phe-Phg-OH
Fmoc-Leu-Ala-OH
Fmoc-Leu-Phg-OH
Fmoc-Leu-Aib-OH
Fmoc-Val-Asp-OH
Fmoc-Met-Glu-OH
Fmoc-Trp-Thr-OH
Z-Phe-Ala-OH
Bfa
Bfy
Bfd
Bfe
Bay
Blf
Bl
q
f
Blq
Buf
Bvf
Bvw
Eba
Ffa
Fff
Bu
Bv
Bv
Eb
Ff
f
w
a
a
f
Ff
Ff
l
Ffl
Ff
v
b
a
b
u
d
e
t
Ffv
Ffb
Fla
Ff
Fl
Fl
Flb
Flu
Fvd
Fme
Fwt
Zfa
Zfy
Zff
Fl
Fv
Fm
Fw
Zf
a
y
f
Zf
Z-Phe-
Z-Phe-Tyr-OH
Zf
Z-Phe-
Z-Phe-Phe-OH
Zf
Z-Phe-
q
l
Zfq
Zfl
Z-Phe-Gln-OH
Zf
Z-Phe-
Z-Phe-Leu-OH
Zf
Z-Phe-
u
y
e
f
Zfu
Zay
Zae
Zuf
Zwb
Zpf
Z-Phe-Aib-OH
Za
Za
Zu
Zw
Zp
Z-Ala-
Z-Ala-Tyr-OH
Z-Ala-
Z-Ala-Glu-OH
Z-Aib-
Z-Aib-Phe-OH
Z-Trp-
b
f
Z-Trp-Phg-OH
Z-Pro-
Z-Pro-Phe-OH
On the basis of these results, we tested the behavior of lysine
hydrochloride (3k) with the mixed anhydride of Boc-Phe- (2Bf),
Fmoc-Val- (2Fv), and Z-Leu- (2Zl) using a 0.5 : 1.0 : 1.0 ratio of 3k :
TBA–OH : 2. As expected, the two amino groups of lysine showed
the same reactivity towards 2 affording the corresponding
isotripeptide 11 (Scheme 4) in 55%–76% overall yield.
In order to exclude any incursion of racemization under our
experimental conditions, Eoc-L-Phg-L-Ala-OH (4Eba) was prepared
by the reaction of N-ethoxycarbonyl-L-phenylglycine (1Eb) with
alanine (3a) and its HPLC–ESI–MS profile as well as 1H and
13C NMR spectra were compared with those of the diastereoiso-
meric mixture of Eoc-DL-Phg-L-Ala-OH (diast-4Eba) obtained from
the reaction of racemic N-ethoxycarbonyl-DL-phenylglycine (rac-
1Eb) with 3a. The HPLC–ESI–MS analysis of diast-4Eba evidenced
two distinct peaks at Rt = 40.1 and 41.3 min of the same area. In
addition, the 1H and 13C NMR spectra showed, with the exception
of the ethoxycarbonyl moiety, two sets of peaks for each proton
and carbon signal, respectively. On the other hand, when the
reaction was carried out with 1Eb, the HPLC–ESI–MS profile of
4Eba pointed out the presence of only one peak at Rt = 41.3 min
and the corresponding 1H and 13C NMR spectra did not exhibit a
similar complexity. The analogous chromatographic and NMR
behaviour found in all the synthesized a-N-protected dipeptides
4 confirmed that the present protocol proceeds with retention
of configuration. It is worth to point out that when we attempted
to synthesize 4Eba following the method proposed by Noguchi
et al. [72], the HPLC–ESI–MS analysis of the crude product
evidenced the presence of a mixture of Eoc-L-Phg-Ala-OH
(4Eba) and Eoc-D-Phg-Ala-OH (epi-4Eba) in the approximate
ratio 80 : 20.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2013; 19: 315–324

SYNTHESIS OF a-N-PROTECTED DIPEPTIDE ACIDS
Table 3. Yields and some properties of a-N-protected dipeptide acids 4 prepared
4
Product
Recrystallization solvent
Yield (%)
mp (ꢀC)
[a]2D0 (c, solvent)
Bfa
Bfy
Bfd
Bfe
Bay
Blf
Boc-Phe-Ala-OH
Boc-Phe-Tyr-OH
Boc-Phe-Asp-OH
Boc-Phe-Glu-OH
Boc-Ala-Tyr-OH
Boc-Leu-Phe-OH
Boc-Leu-Gln-OH
Boc-Aib-Phe-OH
Boc-Val-Phe-OH
Boc-Val-Trp-OH
Eoc-Phg-Ala-OH
Fmoc-Phe-Ala-OH
Fmoc-Phe-Phe-OH
Fmoc-Phe-Leu-OH
Fmoc-Phe-Val-OH
Fmoc-Phe-Phg-OH
Fmoc-Leu-Ala-OH
Fmoc-Leu-Phg-OH
Fmoc-Leu-Aib-OH
Fmoc-Val-Asp-OH
Fmoc-Met-Glu-OH
Fmoc-Trp-Thr-OH
Z-Phe-Ala-OH
Toluene-Hexane
Toluene-Hexane
Toluene-Hexane
Toluene-Hexane
CHCl3-Hexane
CHCl3-Hexane
CHCl3-Hexane
Cyclohexane
81a
124–126
95–97
ꢁ4.7 (0.6, MeOH)
+5.3 (0.8, MeOH)
+4.9 (0.6, MeOH)
ꢁ7.7 (0.9, MeOH)
+6.1 (0.9, MeOH)
ꢁ9.4 (0.8, MeOH)
ꢁ20.1 (0.7, MeOH)
+16.1 (0.6, MeOH)
ꢁ15.5 (0.6, MeOH)
ꢁ5.8 (0.7, MeOH)
ꢁ98.9 (0.8, MeOH)
ꢁ23.6 (0.2, MeOH)d
ꢁ18.0 (0.6, MeOH)
ꢁ24.7 (0.3, MeOH)
ꢁ13.9 (0.5, MeOH)
ꢁ48.6 (0.5, DMSO)
ꢁ28.2 (0.8, MeOH)e
ꢁ42.9 (0.2, MeOH)
ꢁ10.3 (1.0, DMF)f
ꢁ19.7 (0.9, MeOH)
ꢁ18.9 (0.9, MeOH)g
ꢁ13.9 (0.7, MeOH)
ꢁ14.0 (0.5, MeOH)
ꢁ4.4 (0.5, MeOH)
ꢁ17.0 (0.5, MeOH)
ꢁ10.8 (0.6, MeOH)
ꢁ23.5 (0.4, MeOH)
ꢁ8.0 (0.5, MeOH)
+10.7 (0.6, MeOH)
ꢁ17.4 (0.5, MeOH)h
+23.6 (0.5, MeOH)
ꢁ61.8 (0.5, MeOH)
ꢁ13.8 (0.5, MeOH)i
87a
80a
84a; 58b
91a
82a
87a
69a; 79c
70a; 80c
73a; 83c
82a; 75b
81a
78–80
167–169
78–80
120–122
91–93
Blq
Buf
Bvf
Bvw
Eba
Ffa
Fff
135–137
134–136
77–79
CHCl3-Hexane
Toluene-Hexane
CHCl3-Hexane
Toluene-Hexane
Toluene-Hexane
Toluene-Hexane
Toluene-Hexane
Toluene-Hexane
CHCl3-Hexane
CHCl3-Hexane
CHCl3-Hexane
Toluene-Hexane
Toluene-Hexane
CHCl3-Hexane
CHCl3-Hexane
Toluene-EtOAc
CHCl3-Hexane
Toluene-Hexane
Toluene-Hexane
CHCl3-Hexane
Toluene-Hexane
Toluene-EtOAc
Toluene-Hexane
Toluene-Hexane
CHCl3-Hexane
98–100
214–216d
166–168
194–196
221–223
205–207
194–196e
204–206
196–198f
182–184
131–133g
114–116
156–158
189–191
160–162
185–187
139–141
159–161
152–154
143–145h
78–80
86a; 74b
95a
Ffl
Ffv
Ffb
Fla
88a
79a
88a
Flb
Flu
Fvd
Fme
Fwt
Zfa
Zfy
Zff
88a
71a
77a, 59b; 88c
89a; 51b
94a
85a; 74b
84a
91a; 79b
90a
81a
70a
84a; 70b
86a
Z-Phe-Tyr-OH
Z-Phe-Phe-OH
Zfq
Zfl
Z-Phe-Gln-OH
Z-Phe-Leu-OH
Zfu
Zay
Zae
Zuf
Zwb
Zpf
Z-Phe-Aib-OH
Z-Ala-Tyr-OH
Z-Ala-Glu-OH
Z-Aib-Phe-OH
70a; 77c
90a
75a; 80c
Z-Trp-Phg-OH
190–192
132–134i
Z-Pro-Phe-OH
aIsolated yield. The reaction was carried out according to Method B.
bIsolated yield. The reaction was carried out according to Method A.
cIsolated yield. The reaction was carried out according to Method B but using isopropylchloroformate during the synthesis of the mixed
anhydride of 1.
dLit. [55]: mp 208.7–210.6 ꢀC; [a]2D4 ꢁ10.2 (1.5, DMF).
eLit. [55]: mp 179.0–179.8 ꢀC; [a]2D4 ꢁ6.5 (1.5, DMF).
fLit [73]: mp 173–176 ꢀC; [a]2D5 ꢁ10.0 (1.0, DMF).
gLit. [54]: mp 148–150 ꢀC; [a]2D5 ꢁ18.1 (1.5, DMF).
hLit. [54]: mp 121–123 ꢀC; [a]2D5 +0.7 (1.5, DMF).
iLit: [58]: mp 129–130 ꢀC; [a]2D5 ꢁ46.1 (1.0, EtOH).
Finally, in an effort to apply this protocol to the synthesis of
tripeptides 5, we studied the amidation of the mixed anhydride
12 of Fmoc-Leu-Phg-OH (4Flb) with valine (3v). The ESI–MS
analysis of the intact reaction mixture obtained after the formation
(10 min at ꢁ15 ꢀC) of 12 showed the complete conversion of 12
into the corresponding racemization-sensitive 2,4-disubstituted-5
(4H)-oxazolone 13 [78–80]. The HPLC–ESI–MS profile of the
crude product obtained after the reaction of 13 with 3v
evidenced the presence of a mixture of the desired tripeptide
Fmoc-L-Leu-L-Phg-L-Val-OH (5Flbv) and its epimer Fmoc-L-Leu-
D-Phg-L-Val-OH (epi-5Flbv) in the approximate ratio of 91 : 9
(Scheme 5). The reaction carried out using the crude dipeptide
4Flb obtained from the first coupling reaction afforded, after
crystallization from toluene, the pure tripeptide 5Flbv in 63%
overall yield after the two steps. In a similar way were synthesized
the tripeptides Fmoc-Leu-Phe-Val-OH (5Flfv) and Boc-Phe-Val-Ala-
OH (5Bfva) and the tetrapeptide Boc-Leu-Phe-Val-Ala-OH (14) in
68%, 66%, and 48% overall yield, respectively.
Conclusions
We have developed an efficient and simple synthesis of a-
N-protected dipeptide acids 4 by the coupling reaction of the easily
accessible mixed anhydride of an a-N-protected amino acid 2 with
a DMSO solution of the free amino acid 3 and aqueous TBA–OH.
J. Pept. Sci. 2013; 19: 315–324 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

VERARDO AND GORASSINI
R2
H
COOH
R
H
COOH
PG
+
N
O
R1 = iso-Bu
N
O
O
O
R2
COOTBA
1
R
H
R1
O
O
+
7 (7-9% yield)
N
NH2
3-TBA
PG
R
1 = iso-Pr
R2
H
COOH
1
+
N
O
O
8 (3-4% yield)
Scheme 2. Formation of carbamates 7 and 8 when the mixed anhydride of sterically hindered amino acids (Val, Aib, Pro) was used.
O
O
Materials and Methods
R
COOTBA
O
R
H
O
O
+TBA-OH
-H2O
General
N
N
PG
PG
N-Ethoxycarbonyl-L-phenylglycine (1Eb) and N-ethoxycarbonyl-
DL-phenylglycine (rac-1Eb) were prepared by reported procedure
[81]. All solvents and reagents were purchased from Aldrich
Chemical Company and used without further purification. The re-
actions were monitored by ESI–MS in the positive and negative
ion mode with a Finnigan LXQ (linear trap) (Thermo Electron
Corporation, Madison, WI, USA) by simply diluting the intact reac-
tion mixture with methanol and directly infusing the obtained
solution into the ion source with the aid of a syringe pump.
HPLC–ESI–MS analyses were performed with the same instrument
coupled with a Finnigan Surveyor LC Pump Plus (Thermo Electron
Corporation, Madison, WI, USA) and equipped with a Finnigan
Surveyor Autosampler Plus (Thermo Electron Corporation, Madison,
WI, USA). The LC separations were performed on a Hypersil AA-ODS
column (200mmꢂ 2.1mm, 5 mm) from Agilent Technologies
(Spinea, Italy) operating at 30 ꢀC at a flow rate of 0.2ml/min with
the mobile phase composed of (i) water modified with 0.1% (v/v)
of formic acid and (ii) methanol. The eluent was maintained at
O
2
9
Scheme 3. Formation of N-protected-N-isobutyloxycarbonyl amino acid
9 in the presence of excess TBA–OH.
The present protocol has been successfully applied also to
amino acids bearing unprotected hydroxyl and acidic side chains
(H-Thr-OH, H-Tyr-OH, H-Asp-OH, and H-Glu-OH). Under these
same experimental conditions, lysine (3k) reacted with two
molecules of mixed anhydride 2 affording the corresponding
isotripeptide 11. The homogeneous phase reaction proceeds
rapidly and with retention of configuration as evidenced by
HPLC–ESI–MS, 1H and 13C NMR analysis. We are currently
investigating the possibility of suppressing the incursion of
epimerization during the synthesis of polypeptide with the
present mixed anhydride method.
Scheme 4. Synthesis of the isotripeptides 11a-c.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2013; 19: 315–324

SYNTHESIS OF a-N-PROTECTED DIPEPTIDE ACIDS
H
H
N
Ph
N
H
N
O
N
Fmoc O
COOH
Ph
H-Val-OH (3v)
TBA-OH
DMSO
H
H
N
Fmoc
N
N
H
COOH
4Flb
O
Fmoc O
Ph
O
NMM
IBCF
epi-13
epi-5Flbv (9% yield)
H
N
H
Ph
H
N
O
N
N
Ph
H-Val-OH (3v)
TBA-OH
DMSO
H
H
N
Fmoc
13
Fmoc O
N
N
H
COOH
O
O
O
Fmoc O
Ph
O
O
O
12
5Flbv (91% yield)
Scheme 5. Formation of the tripeptide Fmoc-L-Leu-L-Phg-L-Val-OH (5Flbv) and its epimer Fmoc-L-Leu-D-Phg-L-Val-OH (epi-5Flbv) via the racemization-
sensitive 2,4-disubstituted-5(4H)-oxazolone 13.
94% A for the first 20 min after injection then the proportion of sol-
vent B was linearly increased to 50% over a period of 10 min and
kept at this concentration until the end of the run. Infrared spectra
were obtained with a Bruker Vector 22 spectrophotometer (Bruker
Corporation, MA, USA) using the KBr technique for solids and
Synthesis of a-N-protected dipeptide acids 4: Method B.
NMM (0.227 ml, 2.06 mmol) was slowly added to a stirred solution
of a-N-protected amino acid 1 (2.00 mmol) in THF (20 ml) at room
temperature. After 5 min, IBCF (0.267 ml, 2.06 mmol) was slowly
added to the reaction mixture cooled down to ꢁ15 ꢀC and
stirring was continued for 10 min at the same temperature. After
this time, N-methylmorpholinium hydrochloride was filtered off
and washed with THF (2–3 ml). The mother liquor was cooled to
0 ꢀC and a solution of amino acid 3 (2.40 mmol) and 55% aqueous
TBA–OH (1.04 ml, 2.20 mmol) in DMSO (7.0 ml) was added in one
lot and the reaction mixture was vigorous stirred for 30 min at the
same temperature. THF was removed under reduced pressure and
the residue, after acidification to pH 2–3 with 5% HCl, was dissolved
in AcOEt (70 ml) and the organic phase washed with H2O
(2 ꢂ 20 ml), brine (15 ml), and finally dried over Na2SO4. After filtra-
tion and evaporation of the solvent in vacuo, the crude product
was recrystallized from suitable solvents affording the a-N-protected
dipeptide acid 4 in 69%–94% overall yield (Table 3).
1
recorded in the range 4000–400 cmꢁ1. H and 13C NMR spectra
were recorded on a Bruker AC-F 200 spectrometer (Bruker Corpora-
tion, MA, USA) at 200 and 50 MHz, respectively, using DMSO-d6 at
40 ꢀC as solvent. NMR peak locations are reported as d values from
TMS. Some 1H multiplets are characterized by the term app
(apparent): this refers only to their appearance and may be an
oversimplification. Optical rotations were determined on suitable
solutions (g/100 ml) at 20 ꢀC using an AP-300 automatic polarime-
ter purchased from ATAGO (Japan). Elemental analyses were
performed with a Carlo Erba Mod. 1106 elemental analyser (Carlo
Erba Strumentazione, Milan, Italy). Melting points were determined
with an automatic Mettler Mod. FP61 (Mettler Instrumente AG,
Greifensee, CH) melting point apparatus and are not corrected.
The synthesis of Boc-Aib-Phe-OH (4Buf), Boc-Val-Phe-OH (4Bvf),
Boc-Val-Trp-OH (4Bvw), Fmoc-Val-Asp-OH (4Fvd), Z-Aib-Phe-OH
(4Zuf), and Z-Pro-Phe-OH (4Zpf) was also carried out by substitut-
ing IBCF with 1 M solution of isopropyl chloroformate in toluene
(2.06 ml, 2.06 mmol) for the preparation of the mixed anhydride
intermediate of 1Bu, 1Bv, 1Fv, 1Zu and 1Zp, respectively. Method
B was followed with only this variation affording the a-N-protected
dipeptide acid 4 in 70%–88% overall yield (Table 3).
Peptide Synthesis
Synthesis of a-N-protected dipeptide acids 4: Method A.
NMM (0.227 ml, 2.06 mmol) was slowly added to a stirred solution
of a-N-protected amino acid 1 (2.00 mmol) in THF (20 ml) at room
temperature. After 5 min, IBCF (0.267 ml, 2.06 mmol) was slowly
added to the reaction mixture cooled down to ꢁ15 ꢀC and
stirring was continued for 10 min at the same temperature. The
reaction mixture was subsequently warmed up to 0 ꢀC and a
solution (ca. pH 6.5) of amino acid 3 (2.20 mmol), 55% aqueous
TBA–OH (1.04 ml, 2.20 mmol) and 1.4 M KH2PO4 (7.71 ml,
10.80 mmol) was added in one lot. The mixture was allowed to
warm to room temperature under vigorous stirring and, after
additional 1 h, THF was removed under reduced pressure. The
residue was acidified to pH 2–3 with 5% HCl, the solid formed
was dissolved in AcOEt (70 ml) and the organic phase washed
with H2O (2 x 20 ml), brine (15 ml), and finally dried over Na2SO4.
After filtration and evaporation of the solvent in vacuo, the crude
product was recrystallized from suitable solvents affording the
a-N-protected dipeptide acid 4 in 58%–79% overall yield (Table 3).
Synthesis of Fmoc-Leu-Phg-Ala-OH (5Flba), Fmoc-Leu-Phe-Val-OH
(5Flfv), and Boc-Phe-Val-Ala-OH (5Bfva)
NMM (1.03 equiv) was slowly added to a stirred solution of all the
previously synthesized crude a-N-protected dipeptide acid 4
(1.00 equiv) in THF (10 mlmmol-1) at room temperature. After
5 min, IBCF (1.03 equiv) was slowly added to the reaction mixture
cooled down to ꢁ15 ꢀC and stirring was continued for 10min at
the same temperature. After this time, N-methylmorpholinium hy-
drochloride was filtered off and washed with THF (2–3 ml). The
mother liquor was cooled to 0 ꢀC and a solution of amino acid
3 (1.20 equiv) and 55% aqueous TBA–OH (1.10 equiv) in DMSO
(3.5mlmmol-1) was added in one lot and the reaction mixture
J. Pept. Sci. 2013; 19: 315–324 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

VERARDO AND GORASSINI
was vigorous stirred for 30min at the same temperature. THF was
removed under reduced pressure and the residue, after acidifica-
tion to pH 2–3 with 5% HCl, was dissolved in AcOEt (50 mlmmol-1)
and the organic phase washed with H2O (2ꢂ 15 mlmmol-1), brine
(10 mlmmol-1), and finally dried over Na2SO4. After filtration and
evaporation of the solvent in vacuo, the crude product was
recrystallized from toluene affording the a-N-protected tripeptide
acids 5 in 63%–68% overall yield after the two steps.
reduced pressure and the residue, after acidification to pH 2–3 with
5% HCl, was dissolved in AcOEt (90 ml) and the organic phase
washed with H2O (2ꢂ 20 ml), brine (15 ml), and finally dried over
Na2SO4. After filtration and evaporation of the solvent in vacuo,
the crude product was recrystallized from CHCl3-hexane affording
the isotripeptides 11a–c in 55%–76% overall yield.
Acknowledgements
Synthesis of Boc-Leu-Phe-Val-Ala-OH (14).
The authors are grateful to Dr. P. Martinuzzi and Dr. S. Turco for
1
recording H and 13C NMR spectra.
NMM (1.03 equiv) was slowly added to a stirred solution of all the
previously synthesized crude tripeptide Boc-Leu-Phe-Val-OH
(5Blfv, 1.00 equiv) in THF (10 ml mmol-1) at room temperature.
After 5 min, IBCF (1.03 equiv) was slowly added to the reaction mix-
ture cooled down to ꢁ15 ꢀC and stirring was continued for 10 min
at the same temperature. After this time, N-methylmorpholinium
hydrochloride was filtered off and washed with THF (2–3 ml). The
mother liquor was cooled to 0 ꢀC and a solution of alanine
(3a, 1.20 equiv) and 55% aqueous TBA–OH (1.10 equiv) in DMSO
(3.5ml mmol-1) was added in one lot and the reaction mixture
was vigorously stirred for 30 min at the same temperature. THF
was removed under reduced pressure and the residue, after acidifi-
cation to pH 2–3 with 5% HCl, was dissolved in AcOEt (50 mlmmol-1)
and the organic phase washed with H2O (2ꢂ 15 ml mmol-1), brine
(10 ml mmol-1), and finally dried over Na2SO4. After filtration
and evaporation of the solvent in vacuo, the crude product
was recrystallized from toluene affording the tetrapeptide Boc-
Leu-Phe-Val-Ala-OH (14) in 48% overall yield after the three steps.
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Synthesis of N,O-bis-(carbobenzyloxyphenylalanyl)-tyrosine (10).
NMM (0.227 ml, 2.06 mmol) was slowly added to a stirred solution
of Z-Phe-OH (1Zf; 0.548 g, 2.00 mmol) in THF (20 ml) at room
temperature. After 5 min, IBCF (0.267ml, 2.06 mmol) was slowly
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was added in one lot and the reaction mixture was vigorously
stirred for 90 min at room temperature. THF was removed under
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