Comparative investigation of the cleavage step in the synthesis of model peptide resins: Implications for Nα-9- fluorenylmethyloxycarbonyl-solid phase peptide synthesis

Article abstract of DOI:10.1248/cpb.55.468

Based on our studies of the stability of model peptide-resin linkage in acid media, we previously proposed a rule for resin selection and a final cleavage protocol applicable to the Nα-tert-butyloxycarbonyl (Boc)-peptide synthesis strategy. We found that incorrect choices resulted in decreases in the final synthesis yield, which is highly dependent on the peptide sequence, of as high as 30%. The present paper continues along this line of research but examines the Nα-9-fluorenylmethyloxycarbonyl (Fmoc)-synthesis strategy. The vasoactive peptide angiotensin II (AII, DRVYIHPF) and its [Gly ⁸]-AII analogue were selected as model peptide resins. Variations in parameters such as the type of spacer group (linker) between the peptide backbone and the resin, as well as in the final acid cleavage protocol, were evaluated. The same methodology employed for the Boc strategy was used in order to establish rules for selection of the most appropriate linker-resin conjugate or of the peptide cleavage method, depending on the sequence to be assembled. The results obtained after treatment with four cleavage solutions and with four types of linker groups indicate that, irrespective of the circumstance, it is not possible to achieve complete removal of the peptide chains from the resin. Moreover, the Phe-attaching peptide at the C-terminal yielded far less cleavage (50-60%) than that observed with the Gly-bearing sequences at the same position (70-90%). Lastly, the fastest cleavage occurred with reagent K acid treatment and when the peptide was attached to the Wang resin.

Full text of DOI:10.1248/cpb.55.468

468
Notes
Chem. Pharm. Bull. 55(3) 468—470 (2007)
Vol. 55, No. 3
Comparative Investigation of the Cleavage Step in the Synthesis of Model
Peptide Resins: Implications for Na-9-Fluorenylmethyloxycarbonyl-Solid
Phase Peptide Synthesis
Guita Nicolaewsky JUBILUT,a Eduardo Maffud CILLI,b Edson CRUSCA, Jr.,b Elias Horacio SILVA,a
Yoshio OKADA,c and Clovis Ryuichi NAKAIE*,a
a Department of Biophysics, Universidade Federal de São Paulo; Rua 3 de Maio 100, CEP 04044-020, SP, Brazil: b UNESP,
Department of Biochemistry and Chemistry Technology; Rua Prof. Francisco Degni S/N, CEP 14800-900, Araraquara, SP,
Brazil: and c Faculty of Pharmaceutical Sciences, Kobe Gakuin University; Nishi-ku, Kobe 651–2180, Japan.
Received October 7, 2006; accepted December 21, 2006
Based on our studies of the stability of model peptide-resin linkage in acid media, we previously proposed a
rule for resin selection and a final cleavage protocol applicable to the Na-tert-butyloxycarbonyl (Boc)-peptide syn-
thesis strategy. We found that incorrect choices resulted in decreases in the final synthesis yield, which is highly
dependent on the peptide sequence, of as high as 30%. The present paper continues along this line of research
but examines the Na-9-fluorenylmethyloxycarbonyl (Fmoc)-synthesis strategy. The vasoactive peptide angiotensin
II (AII, DRVYIHPF) and its [Gly8]-AII analogue were selected as model peptide resins. Variations in parameters
such as the type of spacer group (linker) between the peptide backbone and the resin, as well as in the final acid
cleavage protocol, were evaluated. The same methodology employed for the Boc strategy was used in order to es-
tablish rules for selection of the most appropriate linker-resin conjugate or of the peptide cleavage method, de-
pending on the sequence to be assembled. The results obtained after treatment with four cleavage solutions and
with four types of linker groups indicate that, irrespective of the circumstance, it is not possible to achieve com-
plete removal of the peptide chains from the resin. Moreover, the Phe-attaching peptide at the C-terminal yielded
far less cleavage (50—60%) than that observed with the Gly-bearing sequences at the same position (70—90%).
Lastly, the fastest cleavage occurred with reagent K acid treatment and when the peptide was attached to the
Wang resin.
Key words peptide synthesis; peptidyl resin; cleavage; linker group
Since its inception,1) the solid-phase peptide synthesis ried out either in anhydrous hydrogen fluoride24) or in a triflu-
(SPPS) methodology has been systematically improved as oromethanesulfonic acid/TFA/thioanisole cocktail,25) should
the result of a wide variety of experimental investigations. be performed, since a significant decrease in the amount of
These efforts have ranged from optimizing the coupling reac- cleaved peptide can occur in this step. The type of the C-ter-
tion itself (through the use of efficient acylating reagents, mi- minal residue and the length of the peptide sequence seem to
crowave irradiation and variations in temperature)2—5) to affect the overall cleavage yield (which, surprisingly, can be
broadening our knowledge of the complex peptide-resin sol- as high as 30%) as a consequence of incomplete final chain
vation process.6—9) Predictably, methods such as nuclear removal accompanied by premature loss from the resin dur-
magnetic resonance10,11) and Fourier transform infrared spec- ing TFA removal of peptide-resins.
troscopy12,13) have also been tested in attempts to further im-
The results of a comparison between benzhydrylamine-resin
prove SPPS. In our case,14—17) we pioneered the application (BHAR)26) and methylbenzhydrylamine-resin (MBHAR),27)
of the electron paramagnetic resonance (EPR) technique, both used for the synthesis of a-carboxamide peptides in the
which is based on the use of a previously developed amino Boc-chemistry, led us to conclude that the latter is the resin
acid-type marker.18,19)
of choice mainly when the resin-bound amino acid is of the
All of these efforts have led to ongoing improvement of hydrophobic type. However, in the presence of a hydrophilic
the SPPS method. Intriguingly, little attention has yet been residue at the C-terminal position, the difference between the
given to the possibility that incomplete cleavage occurs or two aminated resins in terms of their efficiency depends on
that there is premature removal of peptide chains from the the length of the peptide. When the peptide sequence is
solid support. Within this context, we previously proposed longer, BHAR produces higher yields than does MBHAR.
some rules for the selection of resins used in the Na-tert- This is a consequence of the greater stability of the peptide-
butyloxycarbonyl (Boc) chemistry. Our proposal was based resin linkage of the former toward successive TFA treatments
on the stability of model peptide-resin linkages toward exist- in each synthesis cycle.20,21)
ing acid cleavage procedures,20,21) as well as on the degree to
In the present study, we aimed to take a similar approach
which premature peptide chains are removed during trifluo- to establishing rules for the base-labile Na-9-fluorenylmeth-
roacetic acid (TFA)/Boc removal of peptide-resins. The re- yloxycarbonyl (Fmoc)-protecting group synthesis strategy.28)
sults of other studies22,23) have indicated the need for caution Since it is impossible to occur premature peptide chain cleav-
in selecting the type of resin to be used in the synthesis of age from the resin during the Fmoc group removal in piperi-
peptide sequences containing C-terminal residue in either the dine/dimethylformamide (DMF) solution, other parameters
a-carboxamide or the a-carboxyl function. In addition, ap- were varied. The first was the cleavage capacity of different
propriate final cleavage experiments, which are typically car- acid cocktails used routinely for the final peptide cleavage in
∗ To whom correspondence should be addressed. e-mail: clovis@biofis.epm.br
© 2007 Pharmaceutical Society of Japan

March 2007
469
Table 1. Percentage of Peptide Cleavage from the Resin in Different Acid
Solution (at 25 °C, for 2 h)
the Fmoc-synthesis methodology. The second was the type of
linker group used for separating the peptide chain from the
resin matrix. These variations were therefore tested using two
types of peptide sequences that differed in the hydrophobic-
ity of their C-terminal amino acids. The vasoactive an-
giotensin II (AII, DRVYIHPF) and its [Gly8]-AII analogue
were deliberately synthesized using different linker groups
attached to the solid support and designed for the synthesis
of peptides containing carboxamide or carboxyl groups at
their C-terminal position. Besides the Wang resin29) that at-
taches a p-benzyloxybenzyl alcohol spacer to a polystyrene-
type solid support and is used routinely for the synthesis of
peptide acid, the other three tested resins were all character-
ized by containing different linker groups coupled to
MBHAR support. Amongst these, the HMPA resin30) uses
the 4-hydroxymethylphenoxyacetic acid linker (also for the
synthesis of peptide acids) whereas the Knorr31) and Rink32)
resins attach 4-[(R,S)-a-[1-(9H-fluoren-9-yl)-methoxy-form-
amido]-2,4-dimethoxybenzyl-phenoxyacetic acid and 4-
[(2ꢀ,4ꢀ-dimethoxyphenyl) Fmoc-aminomethyl] phenoxyac-
etamido groups, respectively. Among the known acid cock-
tails used for final cleavage, the following solutions were se-
lected28,33): (1): TFA/water (9.5 : 0.5); (2): TFA/p-cresol/water
(9 : 0.5 : 0.5); (3): TFA/ethanedithiol (EDT)/p-cresol/water
(7.5 : 1.5 : 0.5 : 0.5); and (4): TFA/EDT/phenol/thioanisole/
water (8.25 : 0.25 : 0.5 : 0.5 : 0.5, reagent K).
Yield of cleavage (%)
Peptidyl-resin
1a)
2b)
3c)
4d)
[Gly8]AII-Wang-R
[Gly8]AII-Rink-R
[Gly8]AII-HMPA-R
[Gly8]AII-Knorr-R
[Phe8]AII-Wang-R
[Phe8]AII-Rink-R
[Phe8]AII-HMPA-R
[Phe8]AII-Knorr-R
86
79
79
81
64
58
49
59
83
79
70
77
65
45
46
50
74
77
68
75
64
48
47
49
93
80
84
88
76
67
60
69
a) TFA/water (9.5 : 0.5); b) TFA/p-cresol/water (9 : 0.5 : 0.5); c) TFA/EDT/p-
cresol/water (7.5 : 1.5 : 0.5 : 0.5) and d) TFA/EDT/phenol/thioanisole/water
(8.25 : 0.25 : 0.5 : 0.5 : 0.5, reagent K).
Table 1 compares the cleavage yields of AII and [Gly8]-
AII, each submitted to four types of linker resins and to the
TFA cleavage solutions mentioned above. Based on the re-
sults of these experiments, several conclusions were drawn.
First, according to previous results applied to Boc proto-
col,20,21) or even to those obtained in the acid hydrolysis-re- Fig. 1. Time-Course Study of AII Cleavage from Wang, Rink, Knorr and
HMPA-Resins Using Reagent K, at 25 °C
lated investigation of the peptide resins necessary for further
amino acid analysis,34) greater resistance to acid cleavage
was observed with peptides containing Phe at their C-termi- reagent K. Even after 1 h of treatment, approximately 12% of
nal extremities than with those containing Gly at the same lo- this protecting group remained attached to the peptide back-
cation. The use of the former resulted in 50—60% peptide bone, and was only completely removed after 2 h.
removal, compared with 80—90% for the latter. In addition,
Concerning the low cleavage yield of peptide sequences
a higher yield was observed when solution 4 (reagent K) was attaching hydrophobic residues at the C-terminal position,
used. Furthermore, faster removal of peptide chains occurred some recent experiments (not shown) has indicated hat the
when those chains were bound to Wang-resin. Finally, even use of anhydrous HF or TFMSA/TFA/thioanisole treatments
after 2 h of acid treatment, none of peptide resins presented allow, regardless of the type of resin and linker group, a
complete cleavage of peptide chains from their respective cleavage yield of about 95% for this type of peptide se-
solid supports. The mean purity of the cleaved peptides quences. These findings thus suggest that these cleavage
ranged from 70 to 80%, with molecular weights and amino treatments, routinely applied only in Boc chemistry would be
acid compositions that were consistent with the theoretical also applicable to overcome the mentioned chain removal
values.
To determine the time course of the process of peptide
shortcoming in the Fmoc synthesis strategy.
In conclusion, the present study revealed significant varia-
chain cleavage in the most stable AII-resins, the acid treat- tion in the degree to which peptide chains were cleaved from
ment (with reagent K) was extended for up to 6 h at 25 °C the solid support in the Fmoc-peptide chemistry. Similarly to
(Fig. 1). Even after this long cleavage time, only the peptide- what had been observed for the Boc-synthesis strategy, the
Wang support presented near total peptide removal from the amount of peptide removed from the resin is strongly de-
resin. The cleavage values for the peptides attached to other pendent upon the type of linker group, the cleavage solution
supports (Rink or HMPA resins) did not surpass 70%. These and the type of C-terminal residue in the peptide sequence.
findings suggest that considerable caution should be taken in Previous studies have demonstrated that crude peptide purity
the planning of cleavage procedures. A significant (20— is dependent on the type of cleavage cocktail used.28,35) As a
30%) loss in the overall yield can occur during this step, de- complement, the present study demonstrated the critical in-
pending on the type of peptide-resin pair and cleavage pro- fluence of various factors affecting the overall synthesis
cedure. Finally, the AII-HMPA-resin was tested in order yield, especially in terms of incomplete removal of peptide
to evaluate the known high stability of the Arg 2,2,5,7,8- from the solid support, which has been ignored by some au-
pentamethylchroman-6-sulfonyl (Pmc) side-chain group in thors. These losses can be much greater than predicted but

470
Vol. 55, No. 3
nológico (CNPq–National Council for Scientific and Technological Devel-
opment) for the financial support provided. E.M.C. and C.R.N. are recipients
of CNPq research fellowships.
can be avoided by establishing the appropriate combination
of resin type, linker group and cleavage protocol used.
Experimental
References
All Fmoc amino acids were purchased from Advanced Chemtech
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and reagents were acquired from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.)
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residue. In each synthetic cycle, the Na-Fmoc deprotection step was carried
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at 25 °C and cleaved using solvents 1 through 4 (see text). After the cleavage
reaction was complete, the resin was submitted to exhaustive washings with,
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(AcOH)/water, water and MeOH to guarantee the removal of all cleaved
peptides and other by-products of the reaction. After this treatment, small
aliquots of each dried resin were hydrolyzed as previously reported34) for
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resin was compared to the value obtained for the initial peptide-resin pair,
taken as 100%, and checked against the amount of cleaved peptide. To eval-
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Amino Acid Analysis As recently proposed,34) prior to cleavage, all
peptide-resin pairs were hydrolyzed with a mixture of 12 N HCl/propionic
acid for 100 h at 130 °C to guarantee quantitative removal of peptide chains
from the resin. Pyrex tubes with plastic Teflon-coated screw caps (13ꢁ1 cm)
were used for the hydrolyses, and the amino acid analyses were performed in
a Biochrom 20 plus amino acid analyzer (Pharmacia LKB Biochrom Ltd.,
Cambridge, England) to determine the amount of peptide attached to the
resin.
Analytical RP-HPLC The RP-HPLC analyses were carried out in
TFA/acetonitrile gradient using a Waters Associates HPLC system consist-
ing of two 510 HPLC pumps, automated gradient controller, Rheodyne man-
ual injector, 486 UV detector and 746 data module (Waters, Eschborn, Ger-
many). We used Solvent A (0.1% TFA/H2O) and Solvent B (60% acetoni-
trile/0.1% TFA/H2O with a gradient of 5—95% in 30 min) at a flow rate of
1.5 ml/min. A C18 column (0.46ꢁ25 cm, 5 mm particle size and 300 Å pore
size; Vydac, Hesperia, CA, U.S.A.) was employed. Detection was at lꢂ
210 nm.
Liquid Chromatography/Mass Spectrometry The crude lyophilized
peptides were analyzed on a system composed of a Micromass Platform
LCZ Mass Spectrometer (Micromass, Manchester, U.K.), a Waters Alliance
HPLC, a Waters 996 Photodiode Array detector, and a Compaq Workstation.
The peptides were loaded onto a Waters Nova-Pak C18 reverse-phase HPLC
column (2.1ꢁ150 mm, 3.5 mm particle size and 60 Å pore size), using Sol-
vents A (0.1% TFA/H2O) and B (0.1% TFA in CH3CN/H2O) at a flow rate of
0.4 ml/min, detection at 210 nm and a mass range of 500—3930 Daltons.
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Acknowledgments We thank the Fundação de Amparo à Pesquisa do
Estado de São Paulo (Foundation for the Support of Research in the state of
São Paulo) and the Conselho Nacional de Desenvolvimento Científico e Tec-