Combination of silyl carbamate and amino acid fluoride for solid-phase peptide synthesis

Article abstract of DOI:10.1016/S0040-4039(02)00050-3

Diketopiperadine (DKP) formation is an often encountered side reaction in the synthesis of peptide having C-terminal proline ester. A novel strategy for the avoidance of this side reaction was developed, which utilizes Pfp ester of Tsoc-amino acid and Fmo

Full text of DOI:10.1016/S0040-4039(02)00050-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 1515–1518
Combination of silyl carbamate and amino acid fluoride for
solid-phase peptide synthesis†
Kimitoshi Sakamoto,a Yoshiaki Nakaharaa,b and Yukishige Itoa,*
aRIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
bDepartment of Applied Biochemistry, Tokai University, Hiratsuka-shi, Kanagawa 259-1292, Japan
Received 10 December 2001; revised 21 December 2001; accepted 28 December 2001
Abstract—Diketopiperadine (DKP) formation is an often encountered side reaction in the synthesis of peptide having C-terminal
proline ester. A novel strategy for the avoidance of this side reaction was developed, which utilizes Pfp ester of Tsoc-amino acid
and Fmoc-amino acid fluoride as the second and the third amino acid, respectively. This strategy was applied to the synthesis of
37–53 fragment of the b-chain of human chorionic gonadotropin (hCG). © 2002 Elsevier Science Ltd. All rights reserved.
For the synthesis of long chain peptides, a building
block approach is generally more advantageous than
a stepwise one.1 In order to eliminate the potential
risk of epimerization in the course of the block cou-
pling, fragments having C-terminal glycine or pro-
line are usually used as the carboxylic acid com-
ponents, which in turn are prepared by solid-phase
peptide synthesis (SPPS) performed in a stepwise
manner. In that case, a proper choice of the linker is
critical. Peptide fragments should be cleavable from
the resin as a free acid, and the cleavage conditions
are required to be mild enough to preserve all of the
side chain protecting groups. To fulfill these require-
ments, allyl ester-type linkers have been developed;
Pd(0) can achieve removal of which in a facile man-
ner.2
Scheme 1. Competition between acylation and DKP formation.
* Corresponding author. Tel.: +81-48-467-9430; fax: +81-48-462-4680; e-mail: yukito@postman.riken.go.jp
† This work was supported by CREST, Japan Science and Technology Corporation (JST).
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)00050-3

1516
K. Sakamoto et al. / Tetrahedron Letters 43 (2002) 1515–1518
However, in Fmoc-based SPPS, when the C-terminal
amino acid (AA) is linked via a non-bulky ester group
(e.g. benzyl or allyl ester linker), the formation of
diketopiperazine (DKP, path B) often accompanies the
deprotection of the second AA residue and subsequent
acylation (path A), and results in the formation of
peptides lacking C-terminal two AA residues.3 This
problem is particularly serious in the case of peptides
having C-terminal proline (Scheme 1). In our synthetic
studies on the b-subunit 37–53 fragment of human
chorionic gonadotropin (hCG)4 using allyl ester linker,
we encountered this particular problem (vide infra).
Herein, we report a new strategy effective for the
suppression of DKP formation, which utilizes triiso-
propylsilyloxycarbonyl (Tsoc)5 protected AA pen-
tafluorophenyl (Pfp) ester and AA fluoride, as the
second and third AA residue, respectively.
attention to the combination of silyl carbamate and
acid fluoride6 (Scheme 2). When treated with F−, this
system was expected to give peptide with avoidance of
DKP formation, because (1) N-deprotection and
acylation steps are combined, (2) amine generally reacts
rapidly with acid fluoride7 and (3) only a catalytic
amount of F− should be sufficient to keep the reaction
media near-neutral.
This conception was initially tested in solution phase
using allyl linker esterified Fmoc-Pro 12b,c (Scheme 3).
At first, Fmoc removal with t-dodecanethiol (10
equiv.)8 and DBU (0.1 equiv.) in THF gave 2. On the
other hand, Tsoc-Leu-OPfp 3 was prepared from Boc-
Leu-OH in two steps, including esterification and car-
bamate exchange reaction. Namely, Boc-Leu-OH was
treated with Pfp-OH and DCC in AcOEt to afford
Boc-Leu-OPfp, which was subsequently converted to
Since DKP formation derives from the nucleophilic
attack of liberated amine to the C-terminal ester, its
extent would most likely depend upon the duration of
Fmoc deprotection. Therefore, it was inferred that, if
amine is liberated transiently and captured instanta-
neously by an acylating agent, DKP formation could be
avoided. To create such a circumstance, we turned our
the silylcarbamate
3
under modified Ohfune’s
2,6-di-t-butyl-4-methyl-
conditions9
[(i-Pr)3SiOTf,
pyridine/CH2Cl2, rt, 1.5 h, 88% over two steps]. Com-
pound 3 (1.5 equiv.) was then coupled with 2 (THF, rt,
30 min) to give 4 (84%). Subsequent reaction with
Fmoc-Ala-F (1.5 equiv.) was effected with a catalytic
amount (0.1 equiv.) of Bu4NF (CH2Cl2, rt, 45 min).
Scheme 2. Peptide bond formation from silylcarbamate and acid fluoride.
Scheme 3. Preparation of tripeptide in solution phase. (1) DBU (0.1 equiv.), tert-dodecanthiol (10 equiv.)/THF rt, 5 h; (2)
Pfp-OH, DCC/AcOEt, 0°C, 1 h, then rt, 1.5 h; (3) (i-Pr)3SiOTf (1.5 equiv.), 2,6-di-tert-butyl-4-methylpyridine (1.7 equiv.),
CH2Cl2, 0°C, 30 min, then rt, 1 h, 88% over two steps; (4) THF, rt, 30 min, 84%; (5) Fmoc-Ala-F (1.5 equiv.), Bu4NF (0.1
equiv.)/CH2Cl2, rt, 45 min, 84%.

K. Sakamoto et al. / Tetrahedron Letters 43 (2002) 1515–1518
1517
As expected, smooth formation of the desired tripep-
tide 5 was observed, which was isolated in 84% yield,
without any detectable DKP formation. With this
success in hand, our attention was subsequently
turned to the SPPS of the target sequence 13 (Scheme
4).
automated SPPS, carried out by an ABI 431A Pep-
tide Synthesizer with Fmoc-protocol (System software
Ver. 2.0, coupling by HBTU/HOBt, end capping by
Ac2O). After acidic cleavage, the target sequence was
isolated as C-terminal ester 12 in 30% overall yield
after HPLC purification (Inertsil prep-sil, GL Science
Inc.).13 Dramatic improvement is obvious from Fig.
1; HPLC profiles revealed the clean formation of 12
(Rt 25.71 min, m/z 3133 as M+Na+), in stark contrast
with the result obtained by conventional protocol,
which solely relies upon the Fmoc chemistry. In the
latter case, only a trace amount, if any, of the desired
product was formed. The major product (Rt 13.10
min) proved to have m/z 2923 (M+Na+), which corre-
sponds to 37–51 segment and apparently derives from
DKP formation. Removal of the linker was accom-
plished by treatment with Pd(Ph3P)4 (0.2 equiv.) and
PhNHCH3 (10 equiv.) in DMSO (rt, 4 h) to afford
the C-terminal free acid 13 in 70% yield.
Thus, aforementioned 1 was converted to free acid 6
(2.5 equiv. Et3SiH, 50% TFA/CH2Cl2,10 96%) that
was attached to Sieber amide resin11 (Novabiochem
0.52 mmol/g) through activation by HBTU-HOBt-
DIEA in N-methylpyrrolidone (NMP). Resin bound
proline 7 was N-deprotected with 20% piperidine in
NMP. Resultant 8 was reacted with Pfp ester 3 (2
equiv.) in NMP (rt, 30 min). Subsequent coupling
with Fmoc-Ala-F (1.5 equiv.) was successfully carried
out in CH2Cl2 in the presence of Bu4NF (0.1 equiv.)
to give 10, again with no appreciable DKP forma-
tion. Cleavage from the resin by 2% TFA in CH2Cl2
afforded, after chromatographic purification, tripep-
tide 11 in 87% yield.12
In conclusion, we have developed a novel strategy in
SPPS having C-terminal proline, by which DKP for-
mation can be completely suppressed.
To synthesize the complete 37–53 fragment of hCG
b-subunit, resin-bound tripeptide 10 was subjected to
Scheme 4. Solid-phase synthesis of hCG fragment. (1) 50% TFA, Et3SiH (2.5 equiv.)/CH2Cl2, rt, 1.5 h, 95%; (2) HBTU, HOBt,
DIEA/NMP, rt, 1 h; (3) 20% piperidine/NMP; (4) NMP, rt, 30 min; (5) Fmoc-Ala-F (2.0 equiv.), Bu4NF (0.1 equiv.)/CH2Cl2, rt,
2 h; (6) 2% TFA/CH2Cl2; (7) 20% piperidine/NMP; (8) Fmoc-AA, HBTU, HOBt, DIEA/NMP; (9) 20% piperidine/NMP; (10)
MeNHPh (10 equiv.), Pd(PPh3)4 (0.2 equiv.)/DMSO, rt, 4 h.

1518
K. Sakamoto et al. / Tetrahedron Letters 43 (2002) 1515–1518
3426; (e) Lloyd-Williams, P.; Albericio, F.; Giralt, E.
Tetrahedron 1993, 49, 11065–11133; (f) Benz, H. Synthesis
1994, 337–358.
2. (a) Kunz, H.; Dombo, B. Angew. Chem., Int. Ed. Engl.
1988, 27, 711–712; (b) Nakahara, Y.; Ando, S.; Itakura,
M.; Kumabe, N.; Hojo, H.; Ito, Y.; Nakahara, Y. Tetra-
hedron Lett. 2000, 41, 6489–6493; (c) Nakahara, Y.;
Ando, S.; Ito, Y.; Hojo, H.; Nakahara, Y. Biosci. Bio-
tech. Biochem. 2001, 65, 1358–1368.
3. (a) Fields, G. B.; Noble, R. L. Int. J. Pept. Prot. Res.
1990, 35, 161–214; (b) Pedroso, E.; Grandas, A.; de las
Heras, X.; Eritja, R.; Giralt, E. Tetrahedron Lett. 1986,
27, 743–746; (c) Barany, G.; Albericio, F. J. Am. Chem.
Soc. 1985, 107, 4936–4942; (d) Hardy, P. M.; Sheppard,
P. W. J. Chem. Soc., Perkin Trans. 1 1983, 723–729.
4. (a) Pierce, J. G.; Parsons, T. F. Annu. Rev. Biochem.
1981, 50, 465–495; (b) Lapthorn, A. J.; Harris, D. C.;
Littlejohn, A.; Lustbader, J. W.; Canfield, R. E.; Machin,
K. J.; Morgan, F. J.; Issacs, N. W. Nature 1994, 369,
455–461.
5. Lipschutz, B. H.; Papa, P.; Keith, J. M. J. Org. Chem.
1999, 64, 3792–3793.
6. Ito, Y.; Gerz, M.; Nakahara, Y. Tetrahedron Lett. 2000,
41, 1039–1042.
7. Carpino, L. A.; Beyermann, M.; Wenschuh, H.; Bienert,
M. Acc. Chem. Res. 1996, 29, 268–274.
8. Sheppeck, J. E.; Kar, H.; Hong, H. Tetrahedron Lett.
2000, 41, 5329–5333.
Figure 1. HPLC profiles of peptides prepared as shown in
Scheme 4 (A) and conventional method (B).
9. (a) Kisfaludy, L.; Scho¨n, I. Synthesis 1983, 325–332; (b)
Sakaitani, M.; Ohfune, Y. Tetrahedron Lett. 1985, 26,
5543–5546.
10. Mehta, A.; Jaouhari, R.; Benson, T. J.; Douglas, K. T.
Tetrahedron Lett. 1992, 33, 5441–5444.
11. Sieber, P. Tetrahedron Lett. 1987, 28, 2107–2110.
12. Yields were calculated based upon initial loading of the
resin.
13. Inertsil prep-sil (GL Science Inc.), ƒ 10×250 mm, CHCl3/
MeOH 96/4, 2 ml/min, peaks detected at 254 nm.
References
1. (a) Cordonier, N. K.; Albericio, F.; Barany, G. Int. J.
Pept. Prot. Res. 1990, 35, 527–538; (b) Sasaki, T.; Find-
eis, M. A.; Kaiser, E. T. J. Org. Chem. 1991, 56, 3159–
3168; (c) Barlso, K.; Gatos, D.; Schafer, W. Angew.
Chem., Int. Ed. Engl. 1991, 30, 590–593; (d) Hendrix, J.
C.; Lansbury, P. T., Jr. J. Org. Chem. 1992, 57, 3421–