A novel protecting/activating strategy for β-hydroxy acids and its use in convergent peptide synthesis

Article abstract of DOI:10.1021/jo7025788

(Chemical Equation Presented) β-Hydroxy acids were reacted with hexafluoroacetone and carbodiimides to give carboxy-activated six-membered lactones in good yields. On reaction with amines, the corresponding amides were obtained. We demonstrate the following applications of this protecting/ activating strategy: preparation of carboxamides in solution and on solid phase (both normal and reverse mode); recovery and reuse of the excess material in solid-phase synthesis; and convergent solid-phase peptide synthesis (CSPPS) with peptide segments bearing C-terminal Ser or Thr with very low levels of epimerization (<1%, HPLC).

Full text of DOI:10.1021/jo7025788

A Novel Protecting/Activating Strategy for â-Hydroxy Acids and Its
Use in Convergent Peptide Synthesis†
Jan Spengler,*,‡,§ Javier Ru´ız-Rodr´ıguez,‡,§ Francesc Yraola,| Miriam Royo,§,|
Manfred Winter, Klaus Burger, and Fernando Albericio*,‡,§,#
Institute for Research in Biomedicine, Barcelona Science Park, Josep Samitier 1-5, E-08028 Barcelona,
Spain, CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona
Science Park, Josep Samitier 1-5, E-08028 Barcelona, Spain, Combinatorial Chemistry Unit, Barcelona
Science Park, Spain, Department of Organic Chemistry, UniVersity Leipzig, Johannisallee 29,
D-04103 Leipzig, Germany, and Department of Organic Chemistry, UniVersity of Barcelona,
E-08028 Barcelona, Spain
jan.spengler@irbbarcelona.com; albericio@irbbarcelona.com
ReceiVed December 2, 2007
â-Hydroxy acids were reacted with hexafluoroacetone and carbodiimides to give carboxy-activated six-
membered lactones in good yields. On reaction with amines, the corresponding amides were obtained.
We demonstrate the following applications of this protecting/activating strategy: preparation of
carboxamides in solution and on solid phase (both normal and reverse mode); recovery and reuse of the
excess material in solid-phase synthesis; and convergent solid-phase peptide synthesis (CSPPS) with
peptide segments bearing C-terminal Ser or Thr with very low levels of epimerization (<1%, HPLC).
Introduction
react with nucleophiles like amines to give the corresponding
carboxamides 3 (Scheme 1). In addition to the formation of an
amide bond, this approach offers several advantages over
conventional strategies: (i) a very short (minimal) reaction
sequence (2 steps, because carboxyl-activation/OH-protection
and amide formation/OH-deprotection proceed concomitantly);
(ii) site-selectivity in the reaction with dicarboxylic acids like
1 (exclusively 5-membered lactones are formed); (iii) stability
of the activated lactone, thereby allowing isolation, storage, and
recovery of excess in solid-phase peptide synthesis (SPPS); and
(iv) physical properties (good solubility in organic solvents, easy
Amide and peptide bond formation are key reactions in the
synthesis of peptides and other molecules of biological interest.
A large number of reagents for the dehydrative coupling of
carboxylic acids and amines are known.1 However, some
reagents offer additional advantages under certain conditions.
We have recently reported the use of hexafluoroacetone (HFA)
as a bidentate protecting/activating reagent in the synthesis of
R-hydroxy acids and depsipeptides.2 Thus, malic acid 1 reacts
with HFA to give the 5-membered lactone 2. These lactones
† CSPPS, convergent solid-phase peptide synthesis; DCC, dicyclohexylcar-
bodiimide; DCM, dichloromethane; DIC, diisopropylcarbodiimide; DIEA, N,N′-
diisopropyl ethyl amine; DKP, diketopiperazine; DMAP, 4-N,N′-dimethylami-
nopyridine; DMSO, dimethylsulfoxide; DMF, N,N′-dimethylformamide; EDC*HCl,
N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride; Et2O, diethyl-
ether; HFA, hexafluoroacetone; SPPS, solid-phase peptide synthesis; THF,
tetrahydrofuran.
(1) (a) Albericio, F.; Carpino, L. A. In Methods in Enzymology, Solid-
Phase Peptide Synthesis, Vol. 289; Fields, G. B., Ed.; Academic Press:
Orlando, 1997, 104-126. (b) Humphrey, J. M.; Chamberlin, A. R. Chem.
ReV. 1997, 97, 2243-2266. (c) Kaminski, Z. J. Biopolymers 2000, 55, 140-
165. (d) Albericio, F.; Chinchilla, R.; Dodsworth, D. J.; Najera, C. Org.
Prep. Proc. Int. 2001, 33, 203-303. (e) Han, S.-Y.; Kim, Y.-A. Tetrahedron
2004, 60, 2447-2467. (f) Montalbetti, C. A. G. N.; Falque, V. Tetrahedron
2005, 61, 10827-10852. (g) Bode, J. W. Curr. Opin. Drug DiscoVery 2006,
9, 765-775.
‡ Institute for Research in Biomedicine.
§ CIBER-BBN.
| Combinatorial Chemistry Unit.
(2) Spengler, J.; Bo¨ttcher, C.; Albericio, F.; Burger, K. Chem. ReV. 2006,
106, 4728-4746.
University Leipzig.
# University of Barcelona.
10.1021/jo7025788 CCC: $40.75 © 2008 American Chemical Society
Published on Web 02/21/2008
J. Org. Chem. 2008, 73, 2311-2314
2311

Spengler et al.
SCHEME 2. Carbodiimide-Mediated Formation of
SCHEME 1. Site-Selective Derivatization of Malic Acid
Using HFA
6-Membered Lactones
detection by 19F-NMR) of the activated species.3 However, the
application of the HFA-strategy in solid-phase synthesis is
limited to R-hydroxy acids. The corresponding 5-memberd aza-
and thiaheterocycles derived from R-amino and R-mercapto
acids are not generally suited for solid-phase protocols.4 The
â-hydroxy acid unit represents a common structural motif of
naturally occurring compounds. However, on reaction of HFA
with â-hydroxy acid, the formation of the corresponding
6-membered lactones does not proceed spontaneously, in
contrast to 5-membered lactones. Here we discuss the synthesis
and fields of application of the cyclic lactones derived from
HFA and â-hydroxy acids.5
SCHEME 3. Reuse and Recovery of Dioxanone 5a in
Solid-Phase Peptide Synthesis
Results and Discussion
Malic acid amides 3 reacted with HFA to give semiketals 4.
A rapid cyclization to provide 2,2-bis(trifluoromethyl)-1,3-
dioxan-4-ones 5 was achieved by addition of a carbodiimide
(DIC) as carboxy-activating agent.6 The enantiomers 5a and
5b were isolated in 86 and 90% yield from the L- and D-malic
acid amides 3a and 3b, respectively. On reaction of a solution
of 5 in tert-butyl methyl ether with H-Phe-NH2, the depsipep-
tides 6a,b precipitated and were obtained in 77 and 87% yield,
respectively (Scheme 2, i).7 Analogously, the conformationally
more rigid salicylic acid 7 gave lactone 8 and peptide 9 in
comparable yields (Scheme 2, ii).8 In general, we found this
heterocyclization to be a robust protocol that proceeds with
quantitative conversion and gives isolated yields typically
between 70 and 90%. The six-membered activated lactones are,
like their 5-membered counterparts, storable under exclusion
of moisture.
The dioxanones 5 can be used as activated building blocks
in SPPS. Due to their stability, added excess material can be
recovered and reused. Thus, a solution of a 12-fold excess of 5
in dioxane was added to H-Phe-NH-Rink amide-MBHA-resin.
After approximately 4 h, the coupling was complete (negative
ninhydrin test) and the resin was washed with dioxane. The
filtered and evaporated solution was reused twice for couplings
with new portions of resin. The diasteromer 10a (from 5a) was
obtained with >97% HPLC purity in all three runs after
acylation of the 3-hydroxy group with Fmoc-Leu-OH and
cleavage with TFA-H2O (95:5). Comparison with the diastere-
omer 10b (from 5b) by analytical HPLC revealed that no
epimerization occurs. Some 3-hydroxycarboxylic acids, such as
statines, are very expensive, and therefore, the option for easy
recovery of unreacted excess of activated material by simple
filtration can make this protocol attractive (Scheme 3).
The 6-membered lactones were also generated on solid phase.
Resin 11a, obtained from H-Phe-NH-Rink Amide-MBHA-resin
and 2, was rinsed several times with a solution of HFA in THF.9
A 5-fold excess of DIC was then added. After 2 h, the activated
resin 11b was washed (DMF, then DCM) and dried. The
dioxanone 12a was obtained after cleavage with TFA-H2O (95:
5) in >80% HPLC-purity. Samples of resin 11b were treated
with DMF-solutions of 5 equiv of p-methoxy-benzylamine and
(3) Albericio, F.; Burger, K.; Ru´ız-Rodr´ıguez, J.; Spengler, J. Org. Lett.
2005, 7, 597-600.
(4) Albericio, F.; Burger, K.; Cupido, T.; Ruiz, J.; Spengler, J. ArkiVoc
2005, 6, 191-199.
(5) A preliminary account of this work was presented at the 29th
European Peptide Symposium, September 3-6, 2006, Gdansk, Poland. J.
Pept. Sci. 2006, 12, suppl. S, 119.
(6) The semiketal can be observed in the 19F-NMR spectra. Its signal at
-81 ppm (s) disappears and two quartets are observed.
(7) The reactivity of the 6-membered lactones is comparable to that of
the 5-membered lactones. Thus, primary and secondary amides, hydrazides,
and esters can be obtained on reaction with the corresponding nucleophiles.
See ref 2.
(9) Gaseous (anhydrous) hexafluoroacetone is soluble in organic solvents
(DCM < ethyl acetate e THF, ca. 15 g per 100 g). Its solutions can be
stored in a well-closed bottle at -20 °C. At room temperature, a slow
reaction of HFA with THF takes place. CAUTION: hexafluoroacetone is
volatile and very toxic; therefore, all operations must be performed in an
efficient fumehood with properly skin and eye protection.
(8) 8 was prepared from salicylic acid 7 with HFA/DCC in 74% yield.
2312 J. Org. Chem., Vol. 73, No. 6, 2008

NoVel Strategy for â-Hydroxy Acids
SCHEME 4. Synthesis of Amides on Solid-Phase in Reverse
Mode
SCHEME 5. Activation of Ser Requires Special Conditions
SCHEME 6. Protocol for CSPPS Using the HFA-Strategy
H-Leu-NH2, respectively. After 2 h, cleavage and HPLC analysis
showed no presence of 12a, which indicates a rapid nucleophilic
ring-opening of the resin 11b. Products 12b and 12c were
obtained in 81 and 89% HPLC purity, respectively. Because
the dipeptide Mal-Phe-NH2 (from unreacted resin 11a) was not
detected in the products 12a-c, it is suggested that activation
to 11b proceeds quantitatively. These findings demonstrate that
this activating strategy could be useful for reverse-mode solid-
phase synthesis (Scheme 4).10
This protecting/activating strategy, when applied to the
proteinogenic amino acids Ser and Thr, required special
conditions, because the corresponding dioxanones were found
to be sensitive to epimerization. Thus, enantiomerically pure
13 was obtained after dropwise addition of a solution of Boc-
Ser-OH in HFA/THF into a stirred suspension of excess (3
equiv) of EDC*HCl in HFA/THF.11 After 30 min of reaction,
product 13 was extracted with CHCl3, and the organic phase
was washed with 1 N HCl. This crude product was chirally
more stable (ca. 1% epimerization after 48 h standing at rt when
dissolved in dioxane) than the recrystallized 13 (8% epimer-
ization after 12 h at rt, but less than 1% if stored at -20 °C
when dissolved in dioxane).12 We suppose that the HCl traces
from the washing procedure prevent dissociation of the proton
from the chiral center, which bears two electron-withdrawing
substituents. Thus, to prevent epimerization, it is advisable to
perform coupling reactions of HFA-activated compounds de-
rived from 2-amino 3-hydroxy carboxylic acids with the crude
product (Scheme 5).
described above. The activated species 15a was dissolved in a
minimum of dioxane and added to H-Leu-NH-Rink Amide-
ChemMatrix resin (f ) 0.15 mmol/g).13 The coupling required
a considerably longer time than for monomers (8-16 h, to obtain
a negative ninhydrin test). After acetylation of the OH-group
and cleavage from the resin, the peptide Fmoc-Leu-Phe-Ser-
(Ac)-Leu-NH2 16a was obtained with less than 1% epimeriza-
tion.14 Similar results were found for the preparation of Fmoc-
Leu-Phe-Thr(Ac)-Leu-NH2 16c from Fmoc-Leu-Phe-Thr-OH
14c (Scheme 6).
Recently, the “depsipeptide technique” and the use of
pseudoproline building blocks were applied to overcome the
problem of epimerization during coupling of peptide segments
bearing C-terminal Ser and Thr.15 The HFA-strategy addresses
two more challenges associated with CSPPS. First, the trifluo-
romethyl groups significantly enhance the solubility of the
activated peptide in organic solvents. Thus, a higher stationary
concentration at the resin can be achieved. Second, the recovery
and reuse of the excess of activated building block was found
feasible under carefully controlled conditions (demonstrated with
15b, see Supporting Information).16
Finally, peptides of the type Fmoc-Xaa-Leu-Phe-Ser-OH 17
(Xaa represents an amino acid with an unprotected side-chain
The activation protocol developed for Ser can be readily used
in convergent solid-phase peptide synthesis (CSPPS) for the
coupling of peptide segments bearing Ser or Thr in the
C-terminal position. From a practical viewpoint, it is advanta-
geous that HFA and EDC*HCl be used in excess, because
otherwise it would be difficult to deliver stoechiometric amounts,
when couplings are performed on a milligram scale. Thus, the
peptide segment Fmoc-Leu-Phe-Ser-OH 14a was activated as
(13) Garc´ıa-Martin, F.; Quintanar-Audelo, M.; Garc´ıa-Ramos, Y.; Cruz,
L. J.; Furic, R.; Coˆte´, S.; Gravel, C.; Tulla-Puche, J.; Albericio, F. J. Comb.
Chem. 2006, 8, 213-220.
(14) It can be clearly distinguished by HPLC from the diastereomer 16b
(Fmoc-Leu-Phe-DSer(Ac)-Leu-NH2) by HPLC analysis.
(15) (a) Coin, I.; Doelling, R.; Krause, E.; Bienert, M.; Beyermann, M.;
Sferdean, C. D.; Carpino, L. A. J. Org. Chem. 2006, 71, 6171-6177. (b)
Yoshiya, T.; Sohma, Y.; Kimura, T.; Hayashi, Y.; Kiso, Y. Tetrahedron
Lett. 2006, 47, 7905-7909. (c) Keller, M.; Wohr, T.; Dumy, P.; Patiny,
L.; Mutter, M. Chem.-Eur. J. 2000, 6, 4358. (d) Cupido, T.; Tulla-Puche,
J.; Spengler, J.; Albericio, F. Curr. Opin. Drug DiscoVery DeV. 2007, 10,
768-783.
(10) Thieriet, N.; Guibe´, F.; Albericio, F. Org. Lett. 2000, 2, 1815-
1817.
(11) Revealed by the comparison of the HPLC spectra of H-Ser(Ac)-
Leu-NH2 and H-DSer(Ac)-Leu-NH2, which were prepared on solid phase.
When other carbodiimides, like DIC and DCC, or stoechiometric amounts
of EDC*HCl were used, epimerization was found. Similar results were found
for Fmoc-Ser-OH and Boc-Thr-OH. See Supporting information for data.
(12) Chromatography on silica caused >20% epimerization.
(16) The activated peptide segments epimerize slowly in the presence
of the (basic) amino groups of the resin. In an experiment, about 1%
epimerization was found for the first and the second coupling cycle (the
2nd performed with recovered material), but 5% epimerization was found
after the 3rd coupling. Furthermore, diketopiperazine formation diminishes
the content of activated segment.
J. Org. Chem, Vol. 73, No. 6, 2008 2313

Spengler et al.
TABLE 1. Products of CSPPS in the Presence of Several
Functional Groups
ary amide, carboxy, alcoholic and phenolic OH) are tolerated.
No O-acylation was observed during the amide bond formation
in solution or on solid phase. In CSPPS, epimerization of peptide
segments bearing C-terminal Ser or Thr was reduced to <1%
by appropriate choice of solvent and carbodiimide. The stability
of the 6-membered lactones also allows easy recovery of
unreacted excess of activated material in solid-phase protocols.
Experimental Section
Synthesis of 2,2-Bis(trifluoromethyl)-1,3-dioxan-4-ones 5 and
8. The â-hydroxy acid (2 mmol) was stirred in DCM (25 mL) under
an atmosphere of hexafluoroacetone in a flask equipped with a dry
ice condenser and a valve (CAUTION!). In a slight exothermic
reaction, the hydroxy acid goes into solution (for salicylic acid,
THF was used as solvent). Then, 1.1 equiv (2.2 mmol) of
carbodiimide was added. After 1 h stirring, the precipitated urea
was filtered off and the solvent was evaporated. The dioxanones
were obtained analytically pure after flash chromatography or
recrystallization. They are stable when stored under exclusion of
moisture.18
Activation of Peptide Segments Using Hexafluoroacetone
Solution on a Milligram Scale. Typical procedure: The peptide
(0.02 mmol) was dissolved in a solution of HFA in THF (1 mL,
ca. 15 g HFA in 100 mL THF) (CAUTION!). Then, EDC*HCl
(15 mg, 0.08 mmol) was added and suspended by ultrasonification.
After 30 min shaking, the reaction mixture was quenched with 1
N HCl (3 mL) and the product was extracted with chloroform (2
× 1 mL). The organic phase was dried over MgSO4 and evaporated.
The residue can be used directly for solid-phase couplings.
Solution-Phase Protocol for the Preparation of Amides 6 and
9. Typical procedure: H-Phe-NH2 (177 mg, 0.71 mmol) in acetone
(1 mL) was added to a solution of dioxanone (0.65 mmol) in tert-
butyl methyl ether (2 mL). The resulting suspension was stirred
until complete conversion of dioxanone (TLC, approximately 1 h).
tert-Butyl methyl ether (20 mL) was then added, and the suspension
was stirred vigorously for 12 h. The amides were obtained after
filtration and drying.
Solid-Phase Protocol for Coupling and Recovery of 2,2-Bis-
(trifluoromethyl)-1,3-dioxan-4-ones. Typical procedure: The resin
(0.02 mmol) was pre-swollen in dioxane for 30 min. After removal
of the solvent, dioxanone (0.2 mmol) in dioxane (required volume
to cover the resin) was added and left with occasional stirring until
the reaction was complete (negative ninhydrin test). The resin was
then washed with dioxane (0.5 mL) and the filtrates were collected
in a flask. After evaporation of the solvent, the recovered excess
of dioxanone can be reused. Activated peptide segments were
coupled and the excess was recovered analogously. Storage at
-20 °C is recommended.
functionality) were activated and coupled with H-Leu-NH-Rink
Amide-ChemMatrix resin as described above (Scheme 6). Table
1 summarizes the products (Fmoc-Xaa-Leu-Phe-Ser-Leu-NH2
18) obtained after cleavage from the solid support and identified
by HPLC-MS. The coupling proceeded site-selectively in the
presence of unprotected carboxylic groups in the side-chain
(Table 1, 17a to 18a). The formation of semiketals with
alcoholic or phenolic OH groups was reversible by further
aqueous treatment (Table 1, 17b to 18b and 17c to 18c).
Analogously, the sugar-carboxylic acid baicalin 17d was coupled
without any OH-protection scheme on solid phase to give 18d.
The primary carboxamide of Asn (17e) appears to add HFA
because a mixture of desired product 18e and a product 18e′
with a mass 166 units higher was identified. However, 18e′
could not be transformed to 18e by aqueous treatment. The
product 18f obtained from the Arg-containing peptide 17f
suggests that a reaction of the guanidino group takes place with
2 equiv of HFA. The Met-peptide 17g oxidized partially under
the experimental conditions (Table 1, 18g′).17 Thus, this
activation method tolerates unprotected carboxylic acids, alco-
hols, and phenols.
Acknowledgment. We thank Prof. Ayman el Faham (Uni-
versity of Alexandria) for helpful discussion. This study was
supported by the CICYT (BQU 2006-03794), ISCIII (CIBER,
BBN-0074), the Generalitat de Catalunya (2005SGR 00662),
Institute for Research in Biomedicine, and the Barcelona Science
Park.
Conclusions
Here we show that the protection/activation of â-hydroxy
acids with HFA is a robust protocol that can be used for the
site-selective carboxy-derivatization of a wide range of structur-
ally diverse molecules that contain â-hydroxy acids: malic acid,
sugar carboxylic acids, salicylic acid, the amino acids Ser and
Thr, peptides with C-terminal Ser or Thr-residues, and func-
tionalized resins. Several unprotected functional groups (second-
Supporting Information Available: General procedures, com-
pound characterization and copies of NMR and HPLC spectra. This
material is available free of charge via the Internet at http://pubs.
acs.org.
JO7025788
(17) Peptides containing His and Trp gave complex mixtures.
(18) For the preparation of compound 13, see Supporting Information.
2314 J. Org. Chem., Vol. 73, No. 6, 2008