A protocol for racemization-free loading of Fmoc-amino acids to Wang resin

Article abstract of DOI:10.1016/j.tetlet.2008.02.055

Fmoc-amino acids are conveniently and inexpensively linked to Wang resin using benzyloxybenzyl chloride resin without the problems normally associated with the traditional methods of preparation of Fmoc-amino acid loaded Wang resins, namely, low substitution, unacceptable racemization or use of expensive reagents.

Full text of DOI:10.1016/j.tetlet.2008.02.055

Available online at www.sciencedirect.com
Tetrahedron Letters 49 (2008) 2435–2437
A protocol for racemization-free loading of Fmoc-amino
acids to Wang resin
Krishnarao Sandhya, Bhagavathula Ravindranath *
Bharavi Laboratories (P) Ltd, 44/3, Kanakapura Road, Bangalore 560 062, India
Received 12 November 2007; revised 5 February 2008; accepted 10 February 2008
Available online 13 February 2008
Abstract
Fmoc-amino acids are conveniently and inexpensively linked to Wang resin using benzyloxybenzyl chloride resin without the
problems normally associated with the traditional methods of preparation of Fmoc-amino acid loaded Wang resins, namely, low
substitution, unacceptable racemization or use of expensive reagents.
Ó 2008 Elsevier Ltd. All rights reserved.
Since its introduction in 1973,1 the 4-benzyloxybenzyl
alcohol resin (1), popularly known as the Wang resin, has
been used extensively in solid-phase peptide synthesis with
the Fmoc (9-fluorenylmethyloxycarbonyl) protocol.2 The
reasons for its popularity are its stability, low cost and ease
of operation and cleavage. The original and traditional
method of linking the first Fmoc-amino acid to the resin
involves the use of dicyclohexylcarbodiimide and a base,
usually 4-dimethylaminopyridine (DMAP). While this
method has been satisfactory for simple, non-polar amino
acids, unacceptably high levels of racemization are
observed when amino acids such as histidine, cysteine,
methionine, proline and tryptophan are used.3 Except in
the cases of histidine and cysteine, racemization may be
reduced to acceptable levels by using the symmetrical anhy-
dride (5 equiv, or 10 equiv of the Fmoc-amino acid) and
reducing the amount of DMAP used.4 Another well-recog-
nized problem when using DMAP in the above reaction is
the formation of dipeptides.5 To overcome these problems,
several strategies have been reported,6 but another notable
disadvantage of low substitution levels remained. Sieber7
introduced a method that involves the use of a mixed anhy-
dride (5 equiv) of the Fmoc-amino acid and 2,6-dichloro-
benzoic acid. More recently, Blakenmeyer et al.,8
described a method that involves treating the Fmoc-amino
acid with 1-(mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4-tri-
azole (MSTN) and 1-methylimidazole, but the process is
extremely moisture sensitive and expensive. Apart from
the methods involving direct esterification of the primary
alcohol function noted above, several methods have been
introduced to activate the hydroxyl group for linking
to Fmoc-amino acids. Notable among these are the use
of trichloroacetimidate Wang resin9 and the Mitsunobu
reaction,10 using diethyl azodicarboxylate and triphenyl-
phosphine. Apart from the expensive reagents, about
10 equiv of the Fmoc-amino acid is used in most of
the above methods, which makes the processes very
uneconomical.
From the foregoing discussion, it is obvious that the
extreme popularity and utility notwithstanding, there is
still no satisfactory method of loading Fmoc-amino acids
to Wang resin inexpensively and in high loading without
racemization. The more obvious methods of converting
the Wang resin to the corresponding benzyloxybenzyl chlo-
ride11 and bromide12 resins and treating them with the
cesium salts of Fmoc-amino acids have also been examined
but no reliable data is available on the extent of loading
that can be achieved with different Fmoc-amino acids
under different conditions and the methods are not in
common use.
*
Corresponding author. Tel.: +91 80 2666 7742; fax: +91 80 2666 0785.
E-mail address: bharavi@vsnl.com (B. Ravindranath).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.02.055

2436
K. Sandhya, B. Ravindranath / Tetrahedron Letters 49 (2008) 2435–2437
Fmoc-AA
DIPEA + KI
O
O
O-AA-Fmoc
R
1, R = OH
2, R = Cl
3, AA = Amino Acid
Scheme 1.
We, therefore, examined the various ways of bringing
down the cost of loading the Fmoc-amino acids to the
Wang resin, which is an important requirement, particu-
larly in commercial syntheses of peptides. In this
Letter, we present a protocol for obtaining Fmoc-amino
acid-linked Wang resins rapidly, inexpensively and with
near quantitative loading, avoiding the use of expensive
reagents including cesium salts (Scheme 1). Racemization,
even with histidine derivatives is undetectably low. The
method involves converting the benzyloxybenzyl alcohol
group of the Wang resin, 1, into a benzyloxybenzyl chlo-
ride group using thionyl chloride13 and treating the Wang
chloride resin thus obtained with the Fmoc-amino acid
and a non-nucleophilic base such as diisopropylethylamine
(Hunig’s base) in dimethylformamide (DMF) at room tem-
perature; about 10 mol % of potassium iodide is used to
accelerate the reaction. The protocol described in this
paper, which is characterized by its simplicity in compari-
son to the above cited methods, was arrived at after exper-
imenting with several variations of the Fmoc-amino acid,
the solvent, the metal salt, the base and their relative
proportions.
resulted in the recovery (ꢀ75% after purification) of the
unreacted (excess) Fmoc-amino acid that could be reused.
The process was conveniently scaled up to 1 kg level.
In view of the very mild (room temperature) and near-
neutral conditions employed, racemization during the link-
ing process was considered unlikely. Nevertheless, this was
confirmed to be the case by HPLC analysis of the di-
peptide, Fmoc-Val-His(Trt)OH, prepared by standard
protocols.15
The applicability of the above protocol for, (a) synthesis
of peptides, and (b) for linking protected peptides to Wang
resin was demonstrated by preparing Fmoc-Val-PheOH by
standard protocols using Fmoc-Phe-O-Wang resin made
by the present method and linking it again to Wang resin
using the present protocol.16
Acknowledgement
We thank Professor V. Peesapati, JNT University,
Hyderabad, India, for support and encouragement.
References and notes
Typically, the protocol involves swelling the Wang chlo-
ride resin (2, 1 g; ꢀ1 mmol) in DMF (10 ml), reacting it
with the Fmoc-amino acid (3 mmol), potassium iodide
(0.3 mmol) and diisopropyethylamine (3 mmol) under stir-
ring for 16–24 h at room temperature.14 The loading of the
Fmoc-amino acid on the resin, 3, was determined by both
weight gain and by spectrophotometry2 and the results
with different representative Fmoc-amino acids are summa-
rized in Table 1. As can be seen, the loading, even for more
complex Fmoc-amino acids, was much higher than most
of the methods reported in the literature. The first DMF
filtrate on dilution with water (1:10) and acidification
1. Wang, S.-S. J. Am. Chem. Soc. 1973, 95, 1328.
2. Fmoc Solid Phase Peptide Synthesis; Chan, W. C., White, P. D., Eds.;
Oxford University Press: Oxford, 2000; p 346.
3. Atherton, E.; Benoiton, N. L.; Brown, E.; Sheppard, R. C.; Williams,
B. J. J. Chem. Soc., Chem. Commun. 1981, 336.
4. Atherton, E.; Sheppard, R. C. In Solid-Phase Peptide Synthesis: A
Practical Approach; Rickwood, D., Hames, B. D., Eds.; IRL Press:
Oxford, 1989; p 134.
5. Atherton, E.; Logan, C. J.; Sheppard, R. C. J. Chem. Soc., Perkin
Trans. 1 1981, 538.
6. van Nispen, J. W.; Polderdijk, J. P.; Greven, H. M. Recl. Trav. Chim.
Pays-Bas 1985, 104, 99.
7. Sieber, P. Tetrahedron Lett. 1987, 28, 6147.
8. Blakenmeyer, B.; Nimitz, M.; Frank, R. Tetrahedron Lett. 1990, 31,
1701.
Table 1
9. Phoon, C. W.; Oliver, S. F.; Abell, C. Tetrahedron Lett. 1998, 39,
7959.
10. Nouvet, A.; Lamsty, F.; Lazaro, R. Tetrahedron Lett. 1998, 39, 3469.
11. Mergler, M.; Nyfeler, R.; Costeli, J.; Tanner, R. Tetrahedron Lett.
1989, 30, 6745.
Results of loading of different Fmoc-amino acids and peptides by the
described method
Fmoc-amino
Acid
Reaction Fmoc loading by Fmoc loading by
time (h)
weight gain
(mmol/g)
assay
(mmol/g)
12. Corbett, J. W.; Graciani, N. R.; Mousa, S. A.; DeGrado, W. F.
Bioorg. Med. Chem. Lett. 1997, 7, 1371.
Fmoc-Asp(O-tBu)OH 20
0.49
0.49
0.50
0.55
0.57
0.52
0.47
0.51
0.50
0.52
0.60
0.59
0.53
0.48
13. Raju, B.; Kogan, T. P. Tetrahedron Lett. 1997, 38, 4965.
14. The reaction was monitored by withdrawing a few mg of the resin,
washing and drying the resin (see below) and estimating the remaining
chlorine on the resin by the Volhardt method as well as spectro-
photometrically (by Fmoc-release assay). At the end of the reaction,
the resin was filtered and washed successively with DMF (5 ml),
methanol (5 ml), DMF (2 Â 5 ml), water (2 Â 10 ml), methanol
Fmoc-His(Trt)OH
Fmoc-Asn (Trt)OH
Fmoc-MetOH
Fmoc-ValOH
Fmoc-Trp(Boc)OH
Fmoc-Val-PheOH
20
24
16
16
24
20

K. Sandhya, B. Ravindranath / Tetrahedron Letters 49 (2008) 2435–2437
2437
(5 ml), dichloromethane (2 Â 10 ml), and methanol (2 Â 5 ml) and
then dried.
16. Fmoc-Phe-O-Wang resin (0.56 mmol/g), prepared as per the present
protocol was treated with 20% piperidine in DMF for 1 h and the
resultant deprotected Phe-O-Wang resin was added to a solution of
Fmoc-Val-O-1-hydroxy-benzotriazole ester in THF prepared by
standard protocols (Ref. 2). The peptide thus formed was cleaved
from the resin using DCM–TFA (1:1), yielding Fmoc-Val-PheOH,
quantitatively, and in 100% purity (HPLC). The dipeptide was then
linked to Wang chloride resin using the present method, with
satisfactory results (0.48 mmol/g Fmoc-peptide substituion; Table 1).
15. Fmoc-L-His(Trt)-Wang resin prepared according to the above proto-
col was treated with 20% piperidine in DMF to deblock the amino
group and then treated with the ester of Fmoc-L-valine and 1-
hydroxybenzotriazole to obtain Fmoc-Val-His(Trt)O-Wang resin.
The dipeptide, Fmoc-L-Val-L-His(Trt)OH was released from the resin
using DCM–TFA (1:1) and analyzed by HPLC; no peak for Fmoc-L-
Val-D-His(Trt)OH was detectable.