Synthesis of 'difficult' peptides free of aspartimide and related products, using peptoid methodology

Article abstract of DOI:10.1016/j.tetlet.2006.04.074

We developed an efficient, cost effective strategy for Fmoc-based solid phase synthesis of 'difficult' peptides and/or peptides containing Asp/Asn-Gly sequences, free of aspartimide and related products, using a peptoid methodology for the preparation of N-substituted glycines.

Full text of DOI:10.1016/j.tetlet.2006.04.074

Tetrahedron Letters 47 (2006) 4121–4124
Synthesis of ‘difficult’ peptides free of aspartimide and
related products, using peptoid methodology
Sotir Zahariev,a,* Corrado Guarnaccia,a Csaba I. Pongor,a Luca Quaroni,b
c
a
ˇ
ˇ
´
Masa Cemazˇar and Sandor Pongor
aProtein Structure and Function Group, International Centre for Genetic Engineering and Biotechnology, Padriciano 99,
I-34012 Trieste, Italy
bSincrotrone Trieste, Basovizza, I-34012 Trieste, Italy
cUniversity of Queensland, Brisbane 4072, Queensland, Australia
Received 16 March 2006; revised 13 April 2006; accepted 18 April 2006
Abstract—We developed an efficient, cost effective strategy for Fmoc-based solid phase synthesis of ‘difficult’ peptides and/or pep-
tides containing Asp/Asn-Gly sequences, free of aspartimide and related products, using a peptoid methodology for the preparation
of N-substituted glycines.
ꢀ 2006 Elsevier Ltd. All rights reserved.
The formation of aspartimide or aminosuccinimide
(3-amino-pyrrolidine-2,5-dione, Asu) is the first step of
the well-known degradation of aspartic acid/asparagine
containing peptides and proteins at alkaline, neutral and
acidic pH, both in vitro and in vivo1 (Scheme 1). The
reaction is especially prevalent at Asp/Asn-Gly sites,
and results in a variety of rearranged and racemized
products of which the unnatural b-Asp is generally
formed in the largest amount. Asu formation is espe-
cially problematic in Fmoc-tert-based SPSS because
the strong base, such as piperidine, that is used for
deprotection promotes Asu formation.
tection with Hmb7 and analogues8 (Scheme 1). How-
ever, coupling of residues with Hmb-backbone
protection is inefficient, and is often accompanied by
depsipeptide formation.9,10 Hmb-analogues can only
partially resolve these problems.
We recently found that the Dmb amide protecting group
is efficient in preventing secondary structure formation
at Gly-Gly sites, and is orthogonal with respect to stan-
dard Fmoc SPPS.11 In this work we explore the use of
Dmb, Tmb and Nbzl protecting groups (Z) for the syn-
thesis of difficult/Asu-prone peptides, in three different
strategies: (A) Fmoc-Asn/Asp-(Z)Gly-OH dipeptide
building blocks; (B) Fmoc-(Z)Gly-OH monomer build-
ing blocks; and finally, (C) ‘sub-monomeric’ synthesis
of H-(Z)Gly on the resin in which the intermediate
products do not need to be isolated and purified. Strat-
egy C (Scheme 2) is based on the principle of solid phase
peptoid synthesis.14 The first step is the acylation of an
N-terminal amino group of peptide–resin with bromo-
or chloroacetic acid in the presence of a coupling agent,
which is followed by SN2 displacement of the halogen
with excess primary amine.
Asu formation can be diminished by the use of addi-
tives,2 by decreasing the basicity of the deprotection
mixture3 or by applying (instead of OtBu) bulky, alkyl
type protecting groups to the side chain of Asp.4,5 It
can be eliminated by solid phase immobilization via
the amide bond6 or via reversible amide backbone pro-
Keywords: Aspartimide; Backbone protection; Caged peptides; ‘diffi-
cult’ peptides; Peptoid.
*
Corresponding author. Tel.: +39 040 3757341; fax: +39 040
226555; e-mail: sotir@icgeb.org
Alternative solutions were proposed, such as (i) Na-protecting
group,19 and (ii) linkers for SPPS of protected peptides, both
cleavable under almost neutral conditions,20 and finally, (iii) linkers
cleavable with reagents less basic than PIP.21
We tested the new methods on VKDGYI, a well known
model for Asu-formation,9,12 as well as on
H-G1LFGAIAGFIENGWEGMIDG20 GRKKRRQR-
RR30-OH, a peptide containing HA21-20 and HIV-
TAT48-57 sequences, designed to deliver molecules of
0040-4039/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.04.074

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S. Zahariev et al. / Tetrahedron Letters 47 (2006) 4121–4124
Scheme 1.
Scheme 2.
Table 1. Analytical data of backbone protected dipeptide Fmoc-Asx(Y)-(Z)Gly-OH (entries 1–9) and Fmoc-(Dmb)Gly-OH (entry 10, see General
procedure)17
Entry
Asx
Y
Z
Retention timea (min)
Purity (%) crude/purified
MW calculated/ found [M+H]+
1
2
3
4
5
6
7
8
9
10
Asp
Asp
Asp
Asp
Asp
Asp
Asn
Asn
Asn
—
OtBu
OAll
OtBu
OAll
OtBu
OAll
Trt
Trt
Trt
—
Dmb
Dmb
Tmob
Tmob
Nbzl
Nbzl
Dmb
Nbzl
Tmob
Dmb
11.62
11.05
11.64
11.15
11.48
10.63
12.61
12.65
13.30
10.55
92/98.7
95–97/99.0
94/99.1
88–93/99.1
96/99.2
92/99.1
91/99.0
92/98.2
88/98.5
89–96/99.0
618.67/619.6
602.65/603.5
648.70/649.6
632.66/633.5
603.62/604.4
587.19/588.5
803.90/804.8
788.84/789.6
833.92/834.7
447.17/448.4
Dmb: 2,4-Dimethoxybenzyl; Hmb: 2-hydoxy-4-methoxybenzyl; Tmb: 2,4,6-trimethoxybenzyl; Nbzl: 2-nitrobenzyl.
a RPHPLC separation conditions: Zorbax 300 SB 5C18 (150 · 4.6 mm I.D.) column; linear gradient from A = 0.1% TFA in water to B = 0.1% TFA
in MeCN at flow rate 1.5 mL minÀ1 in 15 min. Detection was at 214 nm or ESI-MS.
Table 2. Analytical data of H-Val-Lys-Asx-Gly-Tyr-Leu-NH2/OH peptides prepared by parallel SPPS18 using preformed building blocks (Table 1,
entries 1 or 10) or ‘sub-monomer route’ for the synthesis of aryl substituted N-benzylglycines
Entry Peptide sequence
MW calculated/[M+H]+ Building block or amine used Desired peptidea (purity, %)
1
2
3
4
5
6
7
8
H-VKDGYI-NH2
H-VKD(OAll)GYI-NH2
H-VKDGYI-NH2
H-VKDGYI-NH2
H-VKDGYI-NH2
H-VKDGYI-NH2
H-VKD(OAll)GYI-NH2
H-VKNGYI-NH2
H-VK(Boc)-D(OAll)-(Dmb)G-Y(tBu)-I-OH 1039.6/1040.3
H-VKD(OAll)-GYI-OH 733.4/734.4
H-VK(Boc)-D(OAll)-(Tmb)G-Y(tBu)-I-OH 1069.6/1070.8
H-VKD(OAll)-GYI-OH 733.4/734.4
H-VK(Boc)-D(OtBu)-(Nbzl)GY(tBu)-I-OH 1040.9/1041.8
692.4/693.4
732.4/—
—
—
45b
ndb
94b
692.4/693.4
692.4/693.4
692.4/693.4
692.4/693.4
732.4/733.3
691.4/692.4
Fmoc-(Dmb)G-OH
Fmoc-D(OtBu)-(Hmb)G-OH 92b
Fmoc-D(OtBu)-(Dmb)G-OH 92b
Dmb-NH2
Dmb-NH2
Dmb-NH2
Dmb-NH2
Dmb-NH2
Tmb-NH2
Tmb-NH2
Nbzl-NH2
Nbzl-NH2
Nbzl-NH2
Nbzl-NH2
90b
92b
90b
94c
92c
93c
91c
94c
88c
98c,d
95c
9a
9
10a
10
11a
11
12a
12
H-VKD-(Nbzl)GYI-OH
H-VK(Boc)-N(Trt)-(Nbzl)GY(tBu)-I-OH
H-VKN-(Nbzl)GYI-OH
828.4/829.3
1225.6/1226.5
827.4/828.4
nd—(<1%).
a By RPHPLC after treatment with 20% piperidine in DMF at room temperature for 24 h.12
b Tentagel R RAM resin, Rapp Polymere, 90 lm, 0.22 mmol/g.
c 2-chlorotrityl resin, NovaBiochem, 200–400 lm, 0.25 mmol/g.
d Separation on Zorbax 300 SB 3.5CN column, all other peptides—on Zorbax 300 SB 5C18 column (for more details see Supplementary data).
interest into cells.13 When prepared with standard
Fmoc-SPPS, both peptides showed extensive formation
of Asu (delta mass À 18 Da) and piperidide (delta
mass + 67 Da) as well as the presence of various race-

S. Zahariev et al. / Tetrahedron Letters 47 (2006) 4121–4124
4123
mized and isomerized products that makes product puri-
fication difficult and often impossible.
The use of 2-nitrobenzylamine for bromine displace-
ment in Strategy C is a new method for the preparation
of backbone-caged peptides (Table 2, entry 11–12). It
can also be applied for the synthesis of cyclic peptides
and—combined with orthogonal side chain protected
aspartic acid—for the synthesis of Asp/Asn-derivatives,
for example, glycopeptides15 or side- to side-chain cyclic
peptides.
Table 1 shows the data of preformed building blocks
prepared for Strategy A (1–9) and for Strategy B (10).
Table 2 summarizes the characteristics of VKDGYI as
prepared with the three strategies described above. In
general, the yield and purity of the products reach,
and even exceed, the level obtained with Hmb protec-
tion. We also noted that the removal of the Dmb pro-
tecting group from the dipeptide building blocks by
TFA is ꢀ30% faster than that of Hmb (for more details
see Supplementary data). In addition, ESI-MS showed
that the peptides were free of Asu and piperidide.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tetlet.
2006.04.074.
Finally, we prepared the above mentioned HA2-TAT
peptideà using Strategies A and C. The yield/purity of
the products were comparable with those obtained with
the Hmb-protected dipeptide building block, and both
were found to be free of Asu/piperidides. Both the
preformed building blocks (Strategies A and B) and
the sub-monomeric approach (Strategy C) introduce
an open chain, proline-like structure, which can disrupt
unwanted H-bonds during the synthesis of difficult
peptides. Derivative 10 (Strategy B) is useful for the
synthesis of Xaa-Gly sequences (including Asn/Asp-
Gly) and potentially allows the incorporation of substi-
tuted N-benzylglycine at any point of the synthesis of
‘difficult’ peptides. The ‘sub-monomeric route’ (Strategy
C) is simple and flexible (different amines can be used for
halogen-displacement), does not require separate or
additional synthetic steps and, at the same time, is more
efficient and ꢀ30 times more cost effective than the use
of premade building blocks. With this procedure, the
yields of aryl substituted N-benzylglycine were nearly
quantitative. The new backbone protecting groups used
here were in many respects superior to the commercial
reagents (shelf stable, easily TFA- or photo-cleavable,
orthogonal) and applicable for the synthesis of both
peptide acids and peptide amides.
References and notes
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2. Lauer, J. L.; Fields, C. G.; Fields, G. B. Lett. Pept. Sci.
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3. Wade, J. D.; Mathieu, M. N.; Macris, M.; Tregear, G. W.
Lett. Pept. Sci. 2000, 7107–7112.
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references cited therein.
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8. Offer, J.; Johnson, T.; Quibell, M. Tetrahedron Lett. 1997,
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therein; Meutermans, W. D.; Bourne, G. T.; Golding, S.
W.; Horton, D. A.; Campitelli, M. R.; Craik, D.; Scanlon,
M.; Smythe, M. L. Org. Lett. 2003, 5, 2711–2714.
à Automated peptide synthesis of HA21-20-HIV-TAT48-57
:
First synthesis of peptide H-GLFGAIAGFIENGWEG-
MIDGGRKKRRQRRR-OH containing the sequences of hemagglu-
tinin HA21-20 and HIV-TAT48-57 was assembled on TentaGel S Trt-
Arg(Pbf)-resin (90 lm, substitution 0.2 mmol gÀ1, 0.05 mmol scales,
Fluka) on automated peptide synthesizer SP3 (Protein Technology),
using standard coupling/deprotection cycle.
The second synthesis was carried out in an identical manner, except
that the commercially available building block Fmoc-Asp (OtBu)-
(Hmb)Gly-OH was used to introduce the DG-sequence.
The third synthesis was carried out in an identical manner, except
that the synthesis of H-(Dmb)Gly20-peptide-Ile-resin was prepared by
a two step sub-monomer solid-phase synthesis, using Dmb-NH2 for
bromine displacement11 (see above and the text). Double coupling
was performed from Met17 to N-terminal Gly1 including.
The DCM washed peptide resins were cleaved/deprotected with
TFA/TIPS/H2O (95/2.5/2.5, v/v/v) for 2.5 h, and the peptides were
isolated in the usual manner. The crude peptides were characterized
by RPHPLC (10–55% MeCN in 60 min, 1.5 mL minÀ1) and LC/ESI-
MS (for more details see Supplementary data), as described.11
´
9. Nicolas, E.; Pujades, M.; Bacardit, J.; Giralt, E.; Alberi-
cio, F. Tetrahedron Lett. 1997, 38, 2317–2320.
10. Sampson, W. R.; Patsiouras, H.; Ede, N. J. J. Pept. Sci.
1999, 5, 403–409, and references cited therein.
11. Zahariev, S.; Guarnaccia, C.; Zanuttin, F.; Pintar, A.;
Esposito, G.; Maravic, G.; Krust, B.; Hovanessian, A. G.;
Pongor, S. J. Pept. Sci. 2005, 11, 17–28, and references
cited therein.
12. Mergler, M.; Dick, F.; Sax, B.; Weiler, P.; Vorherr, T.
J. Pept. Sci. 2003, 9, 36–46.
13. Tamm, L. K. Biochim. Biophys. Acta 2003, 1614, 14–
23.

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S. Zahariev et al. / Tetrahedron Letters 47 (2006) 4121–4124
14. Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W.
H. J. Am. Chem. Soc. 1992, 114, 10646–10647; Figliozzi,
G. M.; Goldsmith, R.; Ng, S. C.; Banville, S. C.;
Zuckermann, R. N. Methods Enzymol. 1996, 267, 437–
447; Burkoth, T. S.; Fafarman, A. T.; Charych, D. H.;
Connolly, M. D.; Zuckermann, R. N. J. Am. Chem. Soc.
2003, 125, 8841–8845, and references cited therein.
15. Ende, F., Ph.D., Thesis, University of Hamburg, Ger-
many, 2001.
building blocks, washed with DMF, DCM, stopped and
kept in refrigerator for the next common step. Table 2,
entries 6 to 12: Freshly prepared 2 M solution of bromo-
acetic acid in DMF (1.75 mL) was mixed with 2 M
solution of DIC in DMF (1.75 mL) containing DIEA
(0.05 mL) and then 0.5 mL of the above preactivated
bromoacetic acid was rapidly added to each of H-
Tyr(tBu)-Ile-resins in reactors from entries 6–12. The
peptide resins were mixed for 0.5 h at room temperature,
filtered and washed with DMF (6·). These steps were
repeated one more time. The reactors were charged with
0.5 mL 1 M solution of the corresponding benzylamine
and closed, and the reaction was allowed to proceed at
40 ꢁC for 4 h. After filtration and standard washing
(DMF, 6·), the reactors were disconnected from the
domino block and then to each one was added Fmoc-
Asx(Y)OH/PyBop/HOBt (5 equiv) as a 0.3 M solution in
DMF, containing DIEA (10 equiv). At this point reactors
1–5 were added to the domino block, washed, deprotected
(PIP/DMF, 2 · 5 min) and washed again. The last two
common coupling/deprotection steps were performed
using Fmoc-Lys(Boc)-OH and Fmoc-Val-OH. The resins
were washed with DMF (2·), PrOH-2, DCM, MeCN and
finally with DCM (2·). Single bead ATR Spectromicro-
scopy of peptide-resins confirms the peptide structures (for
more details see Supplementary data). Portions (ꢀ20–
25 mg) from resin 9–12 were deprotected with HFIP
(4 · 0.5 mL, 10 min each one), evaporated to dryness,
dissolved in MeCN (200 lL, each one) and analyzed
(Table 2, entries 9a–12a). Portions (ꢀ20–25 mg) from resin
1–12 were deprotected with TFA/water/TIPS (95/2.5/2.5,
v/v/, 2 mL per each one) for 1 h at room temperature. The
volatile solvents were evaporated at room temperature and
precipitated with ice-cold Et2O, redissolved in 1 M
NaOAc buffer pH 6.6/MeCN (1/1), evaporated (SpeedVac
concentrator) and analyzed (Table 2, entries 1–12) (for
more details see Supplementary data).
16. Krchnak, V.; Padera, V. Bioorg. Med. Chem. Lett. 1998, 8,
3261–3264.
17. General procedure: Preparation of backbone protected
dipeptides (Table 2, entries 1–9) and Fmoc-(Dmb)Gly-
OH, (Table 2, entry 10): All building blocks were prepared
by modification of the published procedure.11 Briefly,
1.2 mmol H-(Z)Gly-OH (for more details see Supplemen-
tary data) in 1 mL DCM were stirred with 2.5–3.0 mmol
N,O-bis(trimethylsilyl)acetamide until dissolved (6–
10 min), then the solution was added to 1 mmol dry
Fmoc-Asx(Y)-OSu (entries 1–9) or Fmoc-OSu (entry 10),
followed by 1 mL DMF. The mixture was stirred until
activated ester (0.5–2 h) was no more detected (TLC,
CHCl3/MeOH/AcOH = 90/9/1). DCM (10 mL) and 10%
citric acid (6 mL) were added and then stirred for 0.5 h.
The bottom phase was separated, and the upper water/
citric acid phase was reextracted with DCM (2 · 5 mL).
The collected DCM phases were washed with water
(6 · 5 mL), dried (Na2SO4), filtered, washed on the filter
with DCM (2 · 3 mL), and the collected solvent was
evaporated to dryness. Crude products (entries 1–10,
Table 1) were purified to homogeneity >98% (RPHPLC)
using column chromatography on silica gel 60 in CHCl3 to
4% MeOH in CHCl3 (for more details see Supplementary
data). The yields of purified products were >75%.
18. The semi-automatic parallel solid-phase organic synthesis of
peptide acids/amides [Table 2] was performed on a
0.025 mmol scale using liquid distribution manifold
(Domino block).16 Twelve 3 mL polypropylene syringes,
equipped with porous polypropylene discs at the bottom,
were used as reactors. All common coupling/deprotection
steps for the preparation of H-Y(tBu)-I-resins were
performed as described.13 Table 2, entries 1–5: Standard
SPPS was performed in reactors 1–5, including the
coupling step of Fmoc-Asp(OtBu/OAll)-OH or selected
´
19. Isidro-Llobet, A.; Guasch-Camell, J.; Alvarez, M.; Alberi-
cio, F. Eur. J. Org. Chem. 2005, 3031–3039.
20. Wagner, M.; Kunz, H. Angew. Chem., Int. Ed. Engl. 2002,
41, 317–321; Wagner, M.; Dziadek, S.; Kunz, H. Chem-
istry 2003, 9, 6018–6030.
´
21. Rabanal, F.; Pastor, J. J.; Nicolas, E.; Albericio, F.;
Giralt, E. Tetrahedron Lett. 2000, 41, 8093–8096.