A one-pot saponification-coupling sequence suitable for C-terminus peptide elongation using lithium carboxylates

Article abstract of DOI:10.1055/s-0034-1378330

An efficient procedure has been developed for the saponification of common peptide esters, followed by straightforward coupling of the lithium carboxylate. Adding some water to the reaction medium gave faster saponification and did not interfere with the coupling reagent. As peptide chemistry constitutes a major application of the amidation reaction, amino acid substrates were chosen for this study, monitoring both yields and epimerization of the peptides obtained. Georg Thieme Verlag Stuttgart New York.

Full text of DOI:10.1055/s-0034-1378330

LETTER
▌1843
letter
A One-Pot Saponification–Coupling Sequence Suitable for C-Terminus Peptide
Elongation Using Lithium Carboxylates
C-Terminus Peptide Elongation using Lithium Carboxylates
Rabah Azzouz, Sylvain Petit, Jean-Baptiste Rouchet, Laurent Bischoff*
Normandie Univ, COBRA, UMR 6014 et FR 3038, Univ Rouen, INSA Rouen, CNRS, IRCOF, 1 rue Tesnière, 76821 Mont Saint Aignan Cedex,
France
Fax +33(235)522962; E-mail: laurent.bischoff@univ-rouen.fr
Received: 08.04.2014; Accepted after revision: 23.05.2014
for easy HPLC monitoring of epimerization and H-Trp-
Abstract: An efficient procedure has been developed for the sapon-
Ot-Bu·HCl was chosen as the model amine. (LLL) and
ification of common peptide esters, followed by straightforward
(LDL) Cbz-Ala-Phe-Trp-Ot-Bu 1a and 1b, respectively,
were prepared as probes (Scheme 1). We expected that a
coupling of the lithium carboxylate. Adding some water to the reac-
tion medium gave faster saponification and did not interfere with
the coupling reagent. As peptide chemistry constitutes a major ap- cationic coupling agent would have a good affinity for the
plication of the amidation reaction, amino acid substrates were cho-
sen for this study, monitoring both yields and epimerization of the
peptides obtained.
carboxylate, and HBTU was first chosen for this reason.
The preliminary results are listed in Table 1. In the pres-
ence of LiOH alone, the reaction was sluggish. Based on
Key words: lithium carboxylate, peptide coupling, amide, amino
acid, epimerization
the known tetracoordination of the lithium cation,3 an ex-
pected four-fold excess of water resulted in rapid saponi-
fication in polar solvents, although resulting in a poor
solubility of the carboxylate (Table 1, entries 1, 3 and 4).
Amino acid and peptide coupling is a very widespread
In dichloromethane, no reaction occurred. Acetonitrile
methodology, and much research in this area has been de-
and THF resulted in formation of a white suspension of
voted to the discovery of new protecting groups and effi-
the lithium carboxylate, whereas DMF and dioxane af-
cient coupling reagents.1 Many different esters have been
forded much better solubilities of this species. DMF gave
employed as masked acids, most commonly methyl, ben-
good yields and short reaction times though, unfortunate-
zyl, tert-butyl and allyl esters. Cleavage of benzyl esters
ly, with higher epimerization of the amino acid carboxyl-
by hydrogenolysis and of tert-butyl esters by acidic treat-
ate during both saponification and coupling processes.
ment generally requires a simple evaporation to yield the
This epimerization in DMF is in agreement with previous
acid, whereas saponification of methyl esters is generally
findings.4 Finally, dioxane appeared to be the best solvent
followed by acidification and extractive workup to obtain
for this study, since epimerization was only observed for
the free acid.
the coupling product, not during the saponification itself.
We wish to report herein a safe, economical and very con-
venient method for a one-pot conversion of small peptide
alkyl esters to their corresponding (n + 1) peptides.
This observation is in agreement with previous findings in
the field of peptidomimetics.5 In the presence of dichloro-
methane, similar results (Table 1, entries 5–7) were ob-
tained, though leading to a heterogeneous medium,
requiring vigorous stirring. Surprisingly, a high molar ex-
cess of water did not lead to hydrolysis of the coupling
agent, and the best conditions consisted of adding enough
water to achieve complete solubilization of the resulting
lithium carboxylate, i.e. 24 molar equivalents (entry 8).
Finally, when this quantity of water was added from the
beginning, the lithium hydroxide remained partially insol-
uble until its consumption and the lithium carboxylate
gave a clear solution. As expected, further addition of wa-
ter was deleterious to the coupling step (entries 9–11).
These conditions, i.e. LiOH (1.2 equiv), dioxane, (24
equiv), water at room temperature for the saponification,
followed by the addition of the amino acid ester hydro-
chloride and the coupling agent (1.5 equiv), were selected
for the next experiments.
The search for racemization-free methods for the saponi-
fication of amino acid alkyl esters, or the release of chiral
auxiliaries2 has shown the superiority of lithium hydrox-
ide over NaOH or KOH. However, these reactions are
generally carried out in THF–water mixtures, and the
amount of water in such reaction media is not compatible
with in situ peptide couplings. Thus, the acid is first recov-
ered in its free acid form by a subsequent acidification.
In search of convenient experimental conditions for a one-
pot saponification and peptide coupling, we first deter-
mined both the appropriate solvent and the minimum
amount of water needed for the saponification. The choice
of solvent was found to be crucial with regards to address-
ing the solubility of both LiOH and the resulting carbox-
ylate, minimizing epimerization, and subsequent
isolation. The dipeptide Cbz-Ala-Phe-OMe was chosen
SYNLETT 2014, 25, 1843–1846
Advanced online publication: 08.07.2014
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DOI: 10.1055/s-0034-1378330; Art ID: st-2014-d0297-l
© Georg Thieme Verlag Stuttgart · New York

1844
R. Azzouz et al.
LETTER
monitoring showed that epimerization occurred during
the activation process, since the still-unreacted lithium
carboxylate kept its chiral integrity. A lower epimeriza-
tion of the activated amino acid was observed with the
milder reagent PyBOP (Table 2, entry 7). The same obser-
vation arose from the use of the uronium salts HBTU and
HATU, however to a lesser extent.
1) LiOH (1.2 equiv),
H2O, solvent, 3 h
Cbz-Ala-Phe-OMe
Cbz-Ala-Phe-Trp-Ot-Bu
1a: (LLL) ; 1b: (LDL)
2) H-Trp-Ot-Bu•HCl (1.1 equiv),
HBTU, DIEA, 4 h
Scheme 1 Optimization of the solvent and water amount
Table 1 Conversions and Diastereomeric Ratio According to the
Interestingly, in classical protocols, all these reagents re-
quire a stoichiometric amount of base in order to deprot-
onate the acid prior to its activation; however this step can
be avoided with lithium carboxylate, limiting the forma-
tion of side-products.
Solvent and Water Amount, using HBTU as the Coupling Agent
Entry Solvent
Equiv
H2O
Conversion dr
(%)b
(1a:1b)
1
2
DMFa
4
4
100
0
90:10
–
CH2Cl2
1) LiOH, H2O (24 equiv), 30 min
Cbz-Ala-Phe-OMe
Cbz-Ala-Phe-Trp-Ot-Bu
3
DMF–CH2Cl2 (1:1)a
dioxanea
4
89
89:11
90:10
91:09
93:7
84:16
92:08
90:10
87:13
–
2) H-Trp-Ot-Bu•HCl (1.1 equiv),
conditions
4
4
98
Scheme 2 Study of the coupling agent
5
dioxane–CH2Cl2 (1:1)a
dioxane–CH2Cl2 (4:1)a
dioxane–CH2Cl2 (1:4)a
dioxane
4
100
100
100
100
82
Table 2 Conversions and Diastereomeric Ratio According to the
Coupling Agent and Base
6
4
7
4
Entry
Coupling
agent
Base
Conversion 1a/1b
8
24
48
72
96
(%)a
ratiob
9
dioxane
1
2
T3P
DIEA
DIEA
DIEA
DIEA
DIEA
DIEA
DIEA
DIEA
DIEA
none
0
0
–
10
11
dioxane
70
DCC
–
dioxane
10
3
EDCI
44
67:33
88:12
78:22
77:23
92:8
92:8
94:6
96:4
a White suspension obtained after saponification.
b Determined by means of HPLC
4
BrOP
100
100
100
100
100
100
100
5
PyBrOP
PyClOP
PyBOP
HBTU
HATU
HATU
6
We further focused on the subsequent coupling step. Sur-
prisingly and to the best of our knowledge, the coupling of
lithium carboxylates has been hardly documented in the
literature, except for an interesting report published re-
cently.6 Only a few examples of such a conversion have
been reported. For instance a coupling with EDC was used
in the preparation of organometallic complexes.7 Another
coupling of lithium carboxylates with aromatic amines
and hydrazines also used EDC, but no experimental de-
tails were given.8 Although the field of coupling agents is
very broad, we selected classical, commercially available
reagents.
7
8
9
10
a Completion of the coupling step measured (HPLC) by complete dis-
appearance of the carboxylate, generally within 2–4 h.
b Diastereomeric ratios were measured by means of HPLC, prior to
purification, in comparison with an authentic sample of 1b.
In the following experiments (Table 2, Scheme 2), we
checked the importance of the cationic character of the re-
agent, as a competition would arise between the carboxyl-
ate and water. Giving support to this hypothesis, neutral
reagents such as DCC or T3P9 resulted in no reaction (Ta-
ble 2, entries 1 and 2). EDCI exhibits a cationic character,
although the charge is not borne by the atom responsible
for the activation. In this case, a moderate 44% yield was
obtained, although with a high degree of epimerization
(Table 2, entry 3). As expected, the best results were ob-
tained with cationic phosphonium or uronium reagents.
For instance, strongly activating halide compounds such
as BrOP, PyBrOP and PyClOP (Table 2, entries 4–6) pro-
vided good yields of coupling products. However in those
cases, significant epimerizations were observed. HPLC
Nevertheless, an additional equivalent of base is still
needed to release the free amine from the amino acid salt.
We initially thought that the base (DIEA) was responsible
for some epimerization. We were therefore pleased to no-
tice that, when using HATU, the reaction ran smoothly
with no additional base. This can be explained by the pres-
ence of the pyridine nitrogen,10 despite its pKA being low-
er than that of the amino group. Under those conditions, a
lower level of epimerization along with good conversion
were obtained (Table 2, entry 10).
In addition, commercially available esters of amino acids
generally consist of methyl, ethyl, allyl or benzyl esters.
Thus, we also checked that they all afforded easy saponi-
fications under the same conditions. N-Cbz phenylalanine
Synlett 2014, 25, 1843–1846
© Georg Thieme Verlag Stuttgart · New York

LETTER
C-Terminus Peptide Elongation using Lithium Carboxylates
1845
esters (Me, Et, Allyl, Bn) were saponified (30 min at r.t.) In most cases, good yields and low levels of epimerization
with LiOH in dioxane in the presence of 24 molar equiv- were obtained. Only the first tripeptide studied (Cbz-Ala-
alents of water, and coupled with H-Trp-Ot-Bu·HCl, Phe-Trp-Ot-Bu) showed noticeable epimerization (96:4
HATU, DIEA (1.5 equiv). Good yields and very low lev- ratio, see Table 2), and, surprisingly, the formation of
els of epimerization (<1%) were observed.
Cbz-Ala-Phe-Val-Leu-OMe gave a similar result (94:6,
entry 7 in Table 3). On the other hand, the tetrapeptide
Boc-Phe-Met-Val-Leu-OMe (Table 3, entry 15) involv-
ing a similar coupling could be prepared with a very good
diastereomeric purity. Various amino acids were tested,
both for saponification (Ala, Phe, Leu, Val, Met) or cou-
pling (Gly, Ala, Leu, Val, Met), as well as unprotected
histidine (Table 3, entry 5); all of them gave satisfactory
results.11
Having these conditions in hand, we first performed the
saponification–coupling sequence with both Cbz-Ala-
Phe-OMe and Boc-Phe-Met-OMe dipeptides with various
amino acids to give tripeptide products (Scheme 3). We
further used the latter tripeptides to carry out subsequent
elongations to tetrapeptides. In each case, no additional
base was used in order to avoid any epimerization. All
diastereomeric ratios were measured by means of HPLC,
prior to purification.
All these observations make this method a very conve-
nient protocol for the straightforward coupling of lithium
carboxylates, following saponification of classical alkyl
esters.11 As we performed this study with peptides, we
could check the chiral integrity of stereogenic centers dur-
ing the process. The compatibility of our method with the
presence of water in the reaction medium is interesting,
since it emphasizes the higher reactivity of the carboxyl-
ate compared to water, towards cationic coupling agents.
Further work in this field may open the way towards C-
terminus elongation of peptides on solid support, as well
as the coupling of unstable or very polar carboxylic acids.
The results obtained are listed in Table 3; yields corre-
spond to isolated yields after purification by flash chroma-
tography. In each case the reaction was run until
completion, generally four hours at room temperature.
1) LiOH (1.2 equiv), H2O (24 equiv), dioxane
Peptide-OMe
Peptide-AA-OMe
2) H-AA-OMe•HCl (1.1 equiv),
HATU (1.4 equiv)
Scheme 3 One-pot, C-terminus peptide elongation
Table 3 C-Terminus Peptide Elongation in One-Pot Reaction
Acknowledgment
Entry Product, compound number
Yield dr (1a:1b)
(%)
(HTU)a
This work has been partially supported by INSA Rouen, Rouen
University, CNRS, Labex SynOrg (ANR-11-LABX-0029) and
Région Haute-Normandie (CRUNCh network). We are grateful to
the Région Haute-Normandie, pôle Chimie-Biologie-Santé for a
grant to Sylvain Petit. Rabah Azzouz is grateful to the FEDER fun-
ding BIOFLUORG.
1
2
Cbz-Ala-Phe-Gly-OMe, 2
Cbz-Ala-Phe-Leu-OMe, 3
Cbz-Ala-Phe-Val-OMe, 4
Cbz-Ala-Phe-Met-OMe, 5
Cbz-Ala-Phe-His-OMe, 6
Cbz-Ala-Phe-Leu-Leu-OMe, 7
Cbz-Ala-Phe-Val-Leu-OMe, 8
Cbz-Ala-Phe-Met-Leu-OMe, 9
Boc-Phe-Met-Gly-OMe, 10
Boc-Phe-Met-Leu-OMe, 11
Boc-Phe-Met-Val-OMe, 12
Boc-Phe-Met-Ala-OMe, 13
86
87
84
79
86
80
90
91
79
96
91
88
> 99:1 (73:27)b
> 99:1 (77:23)b
>99:1 (74:26)b
>99:1 (83:17)
99:1
3
4
Supporting Information Analytical data, copies of NMR
spectra, and experimental preparations of all compounds are avai-
lable online at http://www.thieme-connect.com/products/ejournals/
5
6
>99:1
journal/10.1055/s-00000083.SunogIopitfrmSanrtnuIpgrfoi
m
p
nirtat
7
94:6
8
>99:1
References and Notes
9
>99:1
(1) (a) El-Faham, A.; Albericio, F. Chem. Rev. 2011, 111, 6557.
(b) Montalbetti, C.; Falque, V. Tetrahedron 2005, 61,
10827. (c) Peptides: Chemistry and Biology; Sewald, S.;
Jakubke, H.-D., Eds.; Wiley-VCH Verlag GmbH & Co:
Weinheim, 2002, and references cited therein.
(2) Evans, D. A. Aldrichimica Acta 1982, 15, 23.
(3) Valnot, J. Y.; Maddaluno, J. In The Chemistry of
Organolithium Compounds; Vol. 2; Rappoport, Z.; Marek,
I., Eds.; Wiley: Hoboken, NJ, 2006, 555.
(4) Griehl, C.; Weight, J.; Jeschkeit, H. J. High. Res.
Chromatogr. 1994, 17, 700.
(5) Polyak, F.; Lubell, W. D. J. Org. Chem. 1998, 63, 5937.
(6) Goodreid, J. D.; Duspara, P. A.; Bosch, C.; Batey, R. A.
J. Org. Chem. 2014, 79, 943.
10
11
12
13
14
15
16
98:2 (81:19)b
97:3 (86:14)b
>99:1 (87:13)b
>99:1
Boc-Phe-Met-Leu-Leu-OMe, 14 90
Boc-Phe-Met-Leu-Ala-OMe, 15 80
Boc-Phe-Met-Val-Leu-OMe, 16 76
Boc-Phe-Met-Ala-Leu-OMe, 17 78
>99:1
>99:1
>99:1
a If some epimerization was observed, it only occurred at C-terminus
of the starting peptide.
(7) Achatz, D.; Lang, M. A.; Völkl, A.; Fehlhammer, W. P.;
Beck, W. Z. Anorg. Allg. Chem. 2005, 631, 2339.
(8) Vasudevan, A.; Ji, Z.; Frey, R. R.; Wada, C. K.; Steinman,
D.; Heyman, H. R.; Guo, Y.; Curtin, M. L.; Guo, J.; Li, J.;
Pease, L.; Glaser, K. B.; Marcotte, P. A.; Bouska, J. J.;
b Diastereomeric ratio obtained with HBTU.
© Georg Thieme Verlag Stuttgart · New York
Synlett 2014, 25, 1843–1846

1846
R. Azzouz et al.
LETTER
mmol, 24 molar equiv). Stirring at r.t. and monitoring by
HPLC showed complete saponification and a clear,
homogeneous solution generally after 3 h.
Amino acid H-AA-OMe, HCl (0.9 mmol) and HATU (0.463
g, 1.22 mmol) were added and the mixture was stirred at r.t.
until completion, generally 4 h.
The diastereomeric purity was measured at this stage, before
isolation and purification.
Davidsen, S. K.; Michaelides, M. R. Bioorg. Med. Chem.
Lett. 2003, 13, 3909.
(9) (a) For a review, see: Llanes, Garcıa. A. L. Synlett 2007,
1328. (b) Dunetz, J. R.; Xiang, Y.; Baldwin, A.; Ringling, J.
Org. Lett. 2011, 13, 5048.
(10) (a) Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397.
(b) Carpino, L. A.; El-Faham, A.; Albericio, F. Tetrahedron
Lett. 1994, 35, 2279.
(11) General Procedure for the Formation of Carboxylates
and Subsequent Coupling Reaction: To a solution of Cbz-
Ala-Phe-OMe (0.3 g, 0.81 mmol) in dioxane (2 mL) at r.t.
were added LiOH (25 mg, 1 mmol) and H2O (0.35 mL, 19.44
After concentration in vacuo, the residue was purified by
silica gel chromatography with a mixture of CH2Cl2–EtOAc
as eluent to afford the pure tripeptide as a white solid.
Synlett 2014, 25, 1843–1846
© Georg Thieme Verlag Stuttgart · New York

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