T. Nuijens et al. / Tetrahedron Letters 57 (2016) 3635–3638
3637
it is advised to cap the remaining free alcohol functionalities with a
B
A
benzoyl group using benzoic anhydride and pyridine to avoid side-
O
O
CTC-
reactions.2
+
HO
Fmoc-AA
N
O
+
HO
OH
N
O
N
Unfortunately, the Trt-OCH2COOH method is not compatible
with the acid sensitive CTC resin. Therefore, we also adopted the
protocol of Kodadek and co-workers20 to synthesize Fmoc-pro-
tected glycolic acid (Fmoc-OCH2COOH) in an overall yield of 68%
and an HPLC purity of 98% (Fig. 2B). Although the synthesis of
Fmoc-OCH2COOH requires tert-butyl ester protection/deprotection
steps, the method is compatible with all commonly used SPPS
resins and it is easier to perform the consecutive Cam-ester synthe-
sis in a peptide synthesizer without the use of corrosive TFA.
Alternatively, unprotected glycolic acid was coupled to a H-Leu-
Rink resin, resulting in a resin bound glycolic acid oligomer (Fig. 3).
After alkaline hydrolysis of the ester bonds (1 N NaOH in DMF), the
Cam-ester could be synthesized in the manner described above.
Even though this method is relatively cheap, it is not compatible
with some acid resins, such as the Wang resin.
DiPEA,
CH2Cl2
DMAP,
THF
O
O
O
O
Fmoc-AA
Fmoc-AA
HO
CTC-
TFA,
Fmoc-AA-OH,
DCC/DMAP
5 vol% TIS
O
O
2.5 vol% TFA,
CH2Cl2
Fmoc-AA
O
O
O
CTC-
OH
Resin recycling,
10 vol% SOCl2,
DMF, 120 min
HBTU/Oxyma,
DiPEA
H-Leu-Wang-
Besides loading glycolic acid onto the resin followed by ester
synthesis with an Fmoc-AA-OH, the synthesis could also be per-
formed in a different order. We reasoned that for a strategy com-
patible with all resins and peptide synthesizers, it would be
easier to first assemble the Fmoc-AA-OCH2COOH building blocks
and couple them to a resin or H-AA-resin using the standard cou-
pling reagents as used for subsequent AA elongation steps (Fig. 4A).
Recently, commercially available Fmoc-AA-benzotriazole
derivatives have been described as good starting materials for
the synthesis of Fmoc-AA-esters in the presence of DMAP.21 Using
glycolic acid tert-butyl ester (HOCH2COOtBu), we were able to syn-
thesize the corresponding Fmoc-AA-OCH2COOtBu derivatives in
near quantitative yields. An aqueous washing step and simple sil-
ica filtration proved sufficient to remove the benzotriazole and any
residual Fmoc-AA-OH hydrolytic by-product. After tert-butyl ester
deprotection using 50 vol% TFA in CH2Cl2 and concentration in
vacuo, the desired Fmoc-AA-OCH2COOH building blocks were
obtained. Optionally the tert-butyl esters could be crystallized
prior to tert-butyl ester deprotection, e.g., using CH2Cl2/hexane.
The use of the Fmoc-AA-OCH2COOH derivatives in the SPPS of pep-
tide C-terminal Cam-esters proved to be successful. This method is
applicable to 8 (Ala, Gly, Ile, Leu, Met, Phe, Pro, Val) out of the 20
standard Fmoc-AAs, but not for those containing acid labile side-
chain protecting groups. We investigated the use of benzyl-ester
protected glycolic acid followed by cleavage by hydrogenation
(data not shown). This method is feasible although partial
deprotection of the Fmoc-group is a risk during hydrogenation,
resulting in a troublesome purification.
O
Fmoc-AA
O
Leu-Wang-
N
H
SPPS
Figure 4. Synthesis of peptide C-terminal Cam-Leu-OH esters using Fmoc-AA-
OCH2COOH building blocks. (A) Starting from Fmoc-AA-benzotriazole starting
materials or (B) starting from a CTC resin.
functionality is theoretically possible, we only observed the desired
coupling of the acid. This was proven by the coupling of benzoic
anhydride, mildly acidic cleavage, concentration in vacuo and
NMR analysis. Subsequently, the different Fmoc-AAs were coupled
to the alcohol functionality using DCC/DMAP (45 min, double cou-
pling). After mildly acidic cleavage using 2% TFA in CH2Cl2, washing
with water (3ꢀ) and brine (1ꢀ) and concentration in vacuo, all 20
proteinogenic Fmoc-AA-OCH2COOH building blocks were obtained
in over 90% yield and over 95% purity. Moreover, the CTC resin could
be reactivated using literature procedures22 and used multiple
times without any yield loss after 5 cycles (Fig. 4). The enantiomeric
purity of all Fmoc-AA-OCH2COOH building blocks was determined
(C.A.T. GmbH & Co, see ESI) and proved to be over 99% except for
His, Met, and Ser (97.3%, 98.7%, and 98.7%, respectively). Optionally
these building blocks could be crystallized to remove any D-enan-
tiomer. To show the applicability of the Fmoc-AA-OCH2COOH
building blocks made via the CTC resin method, a peptide library
was synthesized with 20 different amino acids at the C-terminal
position Ac-Asp-Phe-Ser-Lys-Xxx-OCam-Leu-OH. Except for Cys,
all peptides were obtained in good yield (>75%) and excellent purity
(>95%, see ESI). An enzymatic coupling reaction was performed
using Ac-Asp-Phe-Ser-Lys-Leu-OCam-Leu-OH and H-Ala-Leu-Arg-
NH2. The coupling reaction was performed using Omniligase-1, a
peptide ligase discovered by EnzyPep, which is commercially
available from Iris Biotech GmbH. Full conversion of the peptide
Cam-ester was achieved after 60 min to give the product (Ac-Asp-
Phe-Ser-Lys-Leu-Ala-Leu-Arg-NH2, 94 area%) and the hydrolyzed
Cam-ester side-product (Ac-Asp-Phe-Ser-Lys-Leu-OH, 6 area%),
showing the efficiency of peptide Cam-esters for enzymatic ligation
reactions (see ESI).
Finally, we investigated the use of the hyper acid labile CTC resin
for synthesis of the Fmoc-AA-OCH2COOH building blocks (Fig. 4B).
First, unprotected glycolic acid was coupled to the CTC resin using
DIPEA. Although coupling of both the acid and the alcohol
O
O
HBTU,
Oxyma
DiPEA
HO
OH
O
Leu-Rink-
HO
N
H
+
n
O
Leu-Rink-
H
1N NaOH,
DMF
O
O
Fmoc-AA-OH,
DCC/DMAP
O
Fmoc-AA
HO
Leu-Rink-
SPPS
N
H
Leu-Rink-
N
H
Conclusions
Peptide Cam-esters are key building blocks for enzymatic seg-
ment condensation and their efficient synthesis is crucial for the
overall peptide product purity and yield. We have developed
several strategies for the efficient SPPS of peptide Cam-esters.
Figure 3. Synthesis of peptide C-terminal Cam-Leu-NH2 esters using a Rink resin
and glycolic acid oligomerization followed by hydrolysis of the ester bonds.