p-Nitrobenzyloxycarbonyl (pNZ) as a temporary Na-protecting group in orthogonal solid-phase peptide synthesis - Avoiding diketopiperazine and aspartimide formation

Article abstract of DOI:10.1002/ejoc.200500167

p-Nitrobenzyloxycarbonyl (pNZ) was used as a temporary protecting group for α-amino functionalities in solid-phase peptide synthesis. The corresponding derivatives are readily synthesized solids that perform well on solid phase. The pNZ moiety is orthogonal with the most common protecting groups used in peptide chemistry, and is removed under neutral conditions in the presence of catalytic amounts of acid. The use of pNZ derivatives in conjunction with Fmoc chemistry circumvents typical side reactions associated with the use of piperidine, such as DKP and aspartimide formation. The flexibility of pNZ can be exploited for the preparation of libraries of small organic molecules. Wiley-VCH Verlag GmbH & Co. KGaA, 2005.

Full text of DOI:10.1002/ejoc.200500167

FULL PAPER
p-Nitrobenzyloxycarbonyl (pNZ) as a Temporary Nα-Protecting Group in
Orthogonal Solid-Phase Peptide Synthesis – Avoiding Diketopiperazine and
Aspartimide Formation
Albert Isidro-Llobet,[a] Judit Guasch-Camell,[a] Mercedes Álvarez,[a,b] and
Fernando Albericio*[a,c]
Dedicated to Professor Josep Castells on the occasion of his 80th birthday
Keywords: Amino protecting group / Nitro reduction / SnCl2 reduction / PNZ group in SPPS / Combinatorial chemistry /
Side reactions
p-Nitrobenzyloxycarbonyl (pNZ) was used as a temporary
protecting group for α-amino functionalities in solid-phase
peptide synthesis. The corresponding derivatives are readily
synthesized solids that perform well on solid phase. The pNZ
moiety is orthogonal with the most common protecting
groups used in peptide chemistry, and is removed under neu-
tral conditions in the presence of catalytic amounts of acid.
The use of pNZ derivatives in conjunction with Fmoc chemis-
try circumvents typical side reactions associated with the use
of piperidine, such as DKP and aspartimide formation. The
flexibility of pNZ can be exploited for the preparation of li-
braries of small organic molecules.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2005)
former is ultimately removed by trifluoroacetic acid (TFA),
usually 25–50% in CH2Cl2, whereas the latter are removed
by strong acids, such as anhydrous HF or trifluoromethane-
sulfonic acid.[4] Although the Boc-based method has proven
successful for the preparation of large numbers of peptides,
exposure of the peptide chain to TFA during removal of the
Boc group can also cause premature removal of the benzyl
protecting group. Furthermore, certain peptides containing
fragile residues do not survive the relatively harsh acidic
conditions. Finally, HF can be considered a dangerous gas
as it must be stored and used in a special Teflon reactor.
The aforementioned factors have fueled the development of
milder deprotection strategies. Thus, the majority of pep-
tides now produced on solid phase are prepared using the
fluorenylmethoxycarbonyl (Fmoc)–tert-butyl (tBu) orthog-
onal protection strategy.[4–6] In this scheme the Fmoc group
is normally removed with 20% piperidine in DMF, while
the permanent protecting groups are removed by TFA in
the presence of scavengers. Thus, selective deprotection is
Introduction
The solid-phase methodology for the preparation of pep-
tides developed by Bruce R. Merrifield[1] in the late 1950s
and early 1960s was crucial for elucidating the utility of
peptides as important biochemical tools and, more import-
antly, their roles in several therapeutic areas.[2] Currently,
more than 40 peptide-based drugs are on the market, with
four more in registration, 200 in clinical trials, and more
than 400 in advanced preclinical development.[3] Solid-
phase peptide synthesis (SPPS) strategies are characterized
by the lability of both temporary (for the Nα-amine) and
permanent (for side chains and for anchoring the C-terminal
to the solid support through the handle) protecting groups
used.[4] The seminal SPPS strategy proposed by Merrifield
and fine-tuned over time is based on the graduated acid
lability of tert-butyloxycarbonyl (Boc), as a temporary
group, and benzyl-type permanent protecting groups. The
[a] Barcelona Biomedical Research Institute, Barcelona Scientific governed by alternative cleavage mechanisms rather than by
Park, University of Barcelona,
reaction rates. Although this has become the strategy of
Josep Samitier 1–5, 08028 Barcelona, Spain
choice for the preparation of simple peptides, the synthesis
of more complex molecules such as cyclic or branched sys-
tems may require the use of other protecting groups.[7]
Furthermore, the conditions used to remove the Fmoc
group are too harsh for certain cases and may be incompat-
ible with several sequences. The main drawbacks associated
with the use of Fmoc are that both piperidine, which is a
Fax: +34-93-403-71-26
E-mail: albericio@pcb.ub.es
[b] Laboratory of Organic Chemistry, Faculty of Pharmacy, Uni-
versity of Barcelona,
08028 Barcelona, Spain
[c] Department of Organic Chemistry, University of Barcelona,
08028 Barcelona, Spain
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
Eur. J. Org. Chem. 2005, 3031–3039
DOI: 10.1002/ejoc.200500167
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3031

A. Isidro-Llobet, J. Guasch-Camell, M. Álvarez, F. Albericio
FULL PAPER
base and a rather good nucleophile, and the resulting free is the azide method.[12,14] For the case at hand, an opti-
amine can provoke side reactions. Thus, an optimal alterna- mized version of a recently described one-pot protocol
tive to Fmoc for the protection of Nα-amines would be re- based on the azide was used.[13] Thus, pNZ–Cl was allowed
moved under neutral conditions and would leave the re- to react with NaN3, and the resulting pNZ–N3 was directly
sulting amine masked to prevent side reactions. In addition, treated with free amino acids to afford pNZ–amino acids.
the protecting group should be orthogonal with tert-butyl- These derivatives, which were obtained in relatively high
and allyl-based protecting groups as well as Fmoc, among yields (71–94%) and purity, were characterized by HPLC,
1
others, if it is to be used in the synthesis of complex pep- IR, H/13C NMR, and HRMS.
tides or other organic molecules.
Herein, the use of the p-nitrobenzyloxycarbonyl (pNZ)
(Figure 1) group for the temporary protection of α-amines
Solubility of pNZ- and Fmoc-Amino Acids
in an SPPS strategy is reported.
Protected amino acids should be highly soluble, espe-
cially for running reactions at high concentration and if
automated equipment is to be used to dispense the reagents,
a technique often employed for SPPS. In this regard, pNZ-
amino acids demonstrate superior solubility in DMF to
their corresponding Fmoc derivatives (Table 1).
Figure 1. Structure of pNZ protecting group.
Table 1. Solubility comparison of pNZ-aa-OH with Fmoc deriva-
tives.
The pNZ group, which is a carbamate-type protecting
group, was first described by Carpenter and Gish as an al-
ternative to benzyloxycarbonyl (Z).[8] Furthermore, it has
been used for the protection of the ε-amino group of Lys.[9]
The related p-nitrobenzyl (pNB) group has been used to
protect the C-terminal carboxylic group in side-chain an-
choring strategies for the SPS of head-to-tail cyclic pep-
tides,[10] as well as for various functional groups in tradi-
tional organic synthesis.[11]
Amino acid
Solubility in DMF[a] [g/mL]
pNZ-l-Phe-OH
Fmoc-l-Phe-OH
pNZ-l-Gly-OH
Fmoc-l-Gly-OH
pNZ-l-Asp(OtBu)-OH
Fmoc-l-Asp(OtBu)-OH
0.80
0.31
1.33
0.80
1.00
0.67
[a] All derivatives except pNZ-l-Asp(OtBu)-OH show negligible
solubility in CH2Cl2.
Results and Discussion
Preparation of pNZ-Amino Acids
Removal of the pNZ Group
The classical method for the preparation of Nα-carba-
The pNZ group can be removed by catalytic hydrogena-
mate-protected amino acids is via the corresponding chlo- tion as well as other nitro-reducing methods. For the case
roformates using Schotten–Baumann conditions, which can at hand, the first step was reduction of the nitro group to
generate protected dipeptides as side products.[12] Substan- give the p-aminobenzyloxycarbonyl derivative, which suf-
tial quantities of protected dipeptide can lead to the inser- fers spontaneous collapse by 1,6-electron pair shift to afford
tion of an extra amino acid in the final peptide, which is the quinonimine methide and the carbamic acid. Finally,
unacceptable for compounds with therapeutic applications. the carbamic acid decomposes to the corresponding free
Several approaches based on the use of activated formates amine (Figure 2).[15] If this process is carried out in the pres-
have been proposed to minimize this problematic side reac- ence of catalytic amounts of acid, the final product is ob-
tion.[13] An efficient, competitive and inexpensive procedure tained as an ammonium salt.
Figure 2. Mechanism of pNZ group removal.
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Eur. J. Org. Chem. 2005, 3031–3039

pNZ as Nα-Protecting Group in Orthogonal Solid-Phase Peptide Synthesis
FULL PAPER
As shown in Figure 2, the key step in the pNZ removal
pNZ-Phe-Gly-Gly-Leu-NH-Rink-polystyrene resin was
process is the reduction of the nitro group to the corre- used as a model. Removal of the pNZ group was carried
sponding amine. Although the most common method for out under different conditions using both Na2S2O4 and
this transformation is catalytic hydrogention,[16] it is not SnCl2 as reducing agents. The resulting crude peptides were
useful for SPPS. Other common methods are the use of analyzed by HPLC (Table 2).
metals such as Zn,[11b] Fe,[17] or Sn[17] in acidic solutions.
The data outlined in Table 2 illustrate that SnCl2 is a su-
However, the difficulty of working with metals on solid perior reducing agent than Na2S2O4 for the model case. A
phase, as well as the highly acidic conditions required for 6 m SnCl2 solution is more convenient to use than an 8 m
this chemistry, make it problematic for the case at hand. solution, as the latter is supersaturated and some solid
Thus, two reducing reagents envisaged to be compatible SnCl2 may precipitate. HCl (Entry 13) was slightly superior
with SPS, Na2S2O4,[18] and SnCl2,[8b,19,20] were explored.
to Tos-OH (Entry 12) and HOAc (Entry 6), while the per-
formance of the alcohols with a relatively acidic hydrogen
[HFIP (Entry 10) and TFE (Entry 8)] was inferior.
HCl was thus determined to be the most effective acid.
Increasing the concentration of HCl from 1.6 mm (Entry
14) to 64 mm (Entries 15, 16, and 17) did not improve the
rate of deprotection. The presence of phenol does not in-
crease the rate of deprotection or the purity of the final
product. As might be expected, deprotection occurs faster
at high temperature (50 °C) than at room temp. (Entries 18
and 19).
Na2S2O4
Although the effective nitro-reducing agent Na2S2O4 is
typically used under basic conditions,[21] there are several
reports of its application in neutral or nearly neutral me-
dia.[18]
The main drawback associated with the use of Na2S2O4
on solid phase is its solubility. It is highly insoluble in DMF
and other resin-swelling solvents. Furthermore, the re-
duction of nitro groups by Na2S2O4 requires H2O, which is
not a good solvent for most solid supports. In an effort to
overcome the water use, 15-crown-5 was used to solubilize
Na2S2O4. Several attempts at removing the pNZ group of
pNZ-Orn(Boc)-OH in solution using DMF as a solvent
were first carried out using varying amounts of water and
15-crown-5. It was found by TLC (1% HOAc in EtOAc)
that the rate of cleavage increases with the concentration of
H2O and 15-crown-5.
Orthogonality of pNZ-Amino Acids with Fmoc, Boc, and
Alloc Groups
The preparation of complex targets such as cyclic or
branched peptides, as well as those containing chemicaly
fragile moieties, often requires the use of orthogonal pro-
tecting groups.[6,7] To demonstrate the orthogonality of
pNZ-amino acids with common protecting groups, samples
of pNZ-Phe-OH were dissolved in piperidine/DMF (1:4) or
SnCl2
SnCl2 is a good nitro-reducing agent in the presence of TFA (9:1). TLC (1% AcOH in EtOAc) indicated that the
catalytic amounts of acid. It was initially probed with pNZ-amino acids are totally stable to both deprotection
HOAc, but non-carboxylic acids were used in latter probes reagents after 24 h. Likewise, pNZ-Phe-OH was also stable
to prevent acetylation on solid phase.
to Pd(PPh3)4 and phenylsilane in DCM. Furthermore, Boc-
Table 2. Removal of pNZ group from pNZ-Phe-Gly-Gly-Leu-NH-Rink-polystyrene resin.
Entry
Removal conditions[a]
T [ºC]
t [min]
Yield [%][b]
1
2
3
4
5
6
7
8
1 m Na2S2O4 in H2O/AcCN/EtOH (1:1:1)
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
360
60
420
60
420
60
300
60
300
60
300
60
0
11
35
12
13
91
100
68
100
76
100
86
93
98
58
85
97
97
100
1 m Na2S2O4 and 15-Crown-5 in DMF/H2O (9:1)
1 m Na2S2O4 and 15-Crown-5 in DMF/H2O (9:1)
1 m Na2S2O4, 15-Crown-5, and DIEA in DMF/H2O (9:1)
1 m Na2S2O4, 15-Crown-5, and DIEA in DMF/H2O (9:1)
8 m SnCl2, 1.6 mm HOAc, 0.04 m phenol in DMF
8 m SnCl2, 1.6 mm HOAc, 0.04 m phenol in DMF
6 m SnCl2, 0.04 m phenol in DMF/TFE (19:1)
6 m SnCl2, 0.04 m phenol in DMF/TFE (19:1)
6 m SnCl2, 0.04 m phenol in DMF/HFIP (19:1)
6 m SnCl2, 0.04 m phenol in DMF/HFIP (19:1)
6 m SnCl2,1.6 mm TosOH, 0.04 m phenol in DMF
6 m SnCl2,1.6 mm HCl/dioxane, 0.04 m phenol in DMF
6 m SnCl2,1.6 mm HCl/dioxane, 0.04 m phenol in DMF
6 m SnCl2, 64 mm HCl/dioxane in DMF
9
10
11
12
13
14
15
16
17
18
19
60
2×30
2×10
2×20
2×30
2×10
2×20
6 m SnCl2, 64 mm HCl/dioxane in DMF
6 m SnCl2, 64 mm HCl/dioxane in DMF
6 m SnCl2,1.6 mm HCl/dioxane, 0.04 m of phenol in DMF
6 m SnCl2,1.6 mm HCl/dioxane, 0.04 m of phenol in DMF
50 ºC
50 ºC
[a] Experiments were carried out with 10 mg of resin and 0.5 mL of solvent. [b] Yield was calculated by comparing the areas of the
HPLC peaks corresponding to the protected and the deprotected peptides. As the ε of the unprotected peptide should be lower than that
of the protected peptide, the actual yields are higher than those reported.
Eur. J. Org. Chem. 2005, 3031–3039
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A. Isidro-Llobet, J. Guasch-Camell, M. Álvarez, F. Albericio
The synthesis of H-Ala-Leu-Ser-Tyr-Gly-Phe-NH2 [hu-
FULL PAPER
Phe-OH, Fmoc-Phe-OH, Fmoc-Lys(Boc)-OH, and Fmoc-
Orn(Alloc) were stable to a mixture of 6 m SnCl2 and man phospholipase A2 (18–23)] was carried out similarly
1.6 mm HCl/dioxane in DMF for 2 h. Minimal degradation to the procedure outlined above. The pNZ was removed at
was only observed for Boc-Phe-OH after 24 h under these room temperature, and the couplings employed an N-[(1H-
conditions. The aforementioned data hence confirm that 3-oxy-4-azabenzotriazol-1-yl)(dimethylamino)methylene]-
the pNZ group is orthogonal with Boc, Fmoc, and Alloc N-dimethyliminium hexafluorophosphate (HATU)/DIEA
protecting groups.
mediated method. In this case the neutralization step was
omitted. After final acidolytic cleavage and workup the
crude peptide was obtained in good purity as characterized
by HPLC (Figure 3b) and HPLC-MS.
Solid-Phase Peptide Synthesis Using a pNZ/tert-Butyl
Strategy
Using pNZ-amino acids with tert-butyl side chain pro-
tecting groups, Leu-enkephalinamide and human phospho-
lipase A2 (18–23) were synthesized on a Rink-resin.
Use of the pNZ Group to Circumvent Problems Associated
with Fmoc Chemistry
Two parallel syntheses of H-Tyr-Phe-Gly-Gly-Leu-NH2
(Leu-enkephalinamide) were carried out. Removal of the
pNZ group accomplished with 6 m SnCl2, 1.6 mm HCl/di-
oxane in DMF (2×30 min) at room temperature and at
50 °C, followed by extensive washings with DMF (3×30 s),
DMF/H2O (3×30 s), THF/H2O (3×30 s), DMF (3×30 s),
and DCM (3×30 s) to remove excess SnCl2 as well as any
side products from the protecting group. Before coupling
the subsequent pNZ-amino acid, the resin was neutralized
with diisopropylethylamine (DIEA)/CH2Cl2 (1:9).[22] Coup-
lings were performed using an N,N-diisopropylcarbodiim-
ide (DIPCDI) and 1-hydroxybenzotriazole (HOBt) medi-
ated method. After removal of the last pNZ group, free
peptides were released from the resin by acidolytic cleavage
[TFA/H2O/DCM (90:5:5)] and worked up. Both crude
products were obtained in good purity as characterized by
HPLC (Figure 3a) and HPLC-MS.
As mentioned above, the main drawback associated with
the Fmoc strategy is related to the use of piperidine, which
is an excellent nucleophile and a medium-strength base.
Furthermore, after removal of the Fmoc group, the amino
functionality remains a free amine. Both, the piperidine and
the free amine can cause side reactions such as the forma-
tion of diketopiperazines (DKP) and, in the case of Asp-
containing peptides, aspartimides.
DKP Formation
The free amino group of a resin-bound dipeptide can
attack the peptide-resin anchorage intramolecularly to form
cyclic dipeptides or DKPs.[4a,23,24] Thus, the solid-phase
synthesis of C-terminal peptide acids sometimes requires
special protocols for the incorporation of the second and
third amino acids (Figure 4).
Figure 4. Mechanism of DKP formation.
Although DKP formation is governed by various factors,
the side reaction is more severe in Fmoc-based syntheses
than in Boc-based syntheses.[25] First of all, piperidine cata-
lyzes the side reaction,[26] secondly, removal of the Boc
group is carried out in acidic medium, hence the amine re-
mains protonated and incorporation of the third Boc-
amino acid can be done with in situ neutralization, which
minimizes DKP formation.[27] DKP formation in Fmoc
chemistry can be minimized by various approaches, includ-
ing the use of sterically hindered resins such as those based
on trityl (Trt) or tert-butyl (tBu) groups.[28] Likewise, incor-
poration of the second residue with Trt protection, followed
by Trt removal in acidic medium and subsequent incorpora-
tion of the third residue with in situ neutralization, can also
be employed.[29] Under mild conditions, DKP can be also
minimized by incorporation of the second amino acid pro-
tected with the Alloc group in conjunction with a tandem
deprotection–coupling reaction, which is carried out with
Figure 3. HPLC of (a) Leu-enkephaline and (b) the human phos-
pholipase A2 (18–23) (see Exp. Sect. for chromatographic condi-
tions).
3034
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pNZ as Nα-Protecting Group in Orthogonal Solid-Phase Peptide Synthesis
FULL PAPER
the corresponding fluoride derivative and is favored over
A possible solution is a hybrid strategy in which Fmoc is
used to protect all of the residues preceding an Asp, at
DKP formation.[30]
Owing to its facile removal, it was thought that the pNZ which point pNZ is used to protect the Asp and remaining
group would be a good candidate for the protection of the residues. The use of pNZ here would be advantageous as
second amino acid of a growing peptide on solid phase. To its removal does not require the repetitive use of a base, and
demonstrate this methodology, the tripeptide H-Phe-d-Val- the final cleavage and deprotection of the peptide would
Pro-OH was chosen as a model.[31] Thus, pNZ-d-Val-Pro- be carried out with TFA, which is less prone to generate
AB-Leu-aminomethylresin and Fmoc-d-Val-Pro-AB-Leu- aspartimide than the HF normally used in a Boc/Bzl chem-
aminomethylresin were synthesized with Fmoc-Pro-OH istry strategy.[4a]
and pNZ/Fmoc-d-Val-OH, using a Leu residue as internal
reference amino acid (IRAA) to calculate the yield {AB Orn-Asp-Gly-Tyr-Ile-NH2 model since it contains the se-
linker 3-[4-(hydroxymethyl)phenoxy]propionic acid}. quence Asp-Gly-Tyr-Ile, which is considered to generate a
The hybrid strategy was studied using the peptide H-Ala-
=
While 100% of DKP formation was detected after removal large amount of aspartimides.[33b] Two parallel syntheses
of the Fmoc group of the d-Val residue [piperidine/DMF were carried out on a Rink-resin. In the first synthesis,
(2:8)] and incorporation of the Fmoc-Phe-OH, no DKP Fmoc protection was used for Ile, Tyr, and Gly, and pNZ
was observed in the case of the pNZ peptide resin. In this for Asp(OtBu), Orn(Boc), and Ala, whereas the second syn-
case, the pNZ was removed with 6 m SnCl2, 1.6 mm HCl/ thesis used exclusively Fmoc-amino acids. Furthermore, the
dioxane in DMF (2×30 min) at room temperature peptide H-Ala-Orn-Asp(Gly-Tyr-Ile-NH2)-OH[37] was syn-
and after extensive washings, the Fmoc-Phe-OH (5 equiv.) thesized (using Fmoc chemistry) because it is a possible side
was incorporated in the presence of (7-azabenzotriazol-1- product of the aspartimide corresponding to the model pep-
yloxy)tris(pyrrolidino)phosphonium hexafluorophosphate tide. Fmoc and pNZ groups were removed with 4 h treat-
(PyAOP) (5 equiv.) and DIEA (10 equiv.) using in situ neu- ments of 20% piperidine/DMF or 6 m SnCl2/DMF, respec-
tralization.[32]
tively. While both piperidides were detected in the synthesis
using exclusively Fmoc-protection, neither aspartimide nor
β-peptide were observed in the synthesis using the Fmoc-
pNZ hybrid strategy.
Aspartimide Formation
The cyclization of Asp residues to form aspartimides is
a common side reaction in peptide synthesis.[4a,33] Although
this side reaction is favored under various conditions
(strong acids, excess coupling reagents or basic medium)
Conclusions
This work has demonstrated the benefits of using pNZ
and is sequence-dependent, it is most relevant to Fmoc- as a temporary protecting group for the α-amines in SPPS.
based syntheses due to the repetitive use of piperidine (Fig- The corresponding derivatives are readily synthesized and,
ure 5). Hence, Fmoc strategies not only yield the aspartim- in contrast to other α-amino protecting groups such as Trt
ide-containing peptide but also generate the corresponding and Alloc, they are solids. pNZ is removed under simple,
α- and β-peptides (by hydrolysis of α-imides) and α- and β- neutral conditions in the presence of catalytic amounts of
piperidides (via attack of the aspartimide by piperidine).
acid. pNZ is orthogonal to the most common SPPS pro-
The side reaction is more severe when the β-carboxyl tecting groups such as tBu/Boc, Fmoc, and Alloc. In ad-
group of the Asp is protected with the allyl group, since it dition to its utility for the total elongation of a peptide
forms a better leaving group than tert-butyl.[34] Although chain, the pNZ group can be used in conjuction with Fmoc
the side reaction can be minimized to some extent through chemistry to overcome side reactions associated with the
the use of hindered protecting groups[35] for the Asp and by use of piperidine, such as DKP and aspartimide formation.
protecting the preceeding amide,[36] a more convenient and It is also predicted that the use of pNZ for the preparation
general method is needed.
of C-terminal Cys peptides would circumvent the formation
Figure 5. Mechanism of aspartimide formation with typical side products shown.
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Eur. J. Org. Chem. 2005, 3031–3039
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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A. Isidro-Llobet, J. Guasch-Camell, M. Álvarez, F. Albericio
FULL PAPER
pNZ-Ser(tBu)-OH: M.p. 92.8–93.8 °C. HPLC: Rt = 11.28 min
of N-piperidylalanine, a frequent side reaction when Fmoc
protection is used.[38] The flexibility of pNZ can be ex-
ploited for the preparation of libraries of small organic
molecules.
(Gradient A). IR (KBr): ν = 3364, 3170, 2980, 1741, 1697, 1525,
˜
1
1347 cm–1. H NMR (400 MHz, DMSO): δ = 8.21 (d, J = 8.6 Hz,
2 H), 7.61 (d, J = 8.6 Hz, 2 H), 7.45 (d, J = 8.3 Hz, NH), 5.18 (s,
2 H), 4.10 (m, 1 H), 3,55 (m, 2 H), 1.09 (s, 9 H) ppm. 13C NMR
(100 MHz, DMSO): δ = 172.44 (C), 156.42 (C), 147.60 (C), 145.71
(C), 128.78 (CH), 124.15 (CH), 73.50 (C), 64.93 (CH2), 62.03
(CH2), 55.61 (CH), 27.84 (CH3) ppm. HRMS (CI): m/z calcd. for
C15H21N2O7 [M + H+] 341.1350, found 341.1359.
Experimental Section
General Procedures: Commercial-grade reagents and solvents were
used without further purification. Resins, linkers and amino acid
derivatives, HOBt, DIPCDI, PyAOP, HATU, p-nitrobenzyl chloro-
formate and sodium azide were obtained from NovaBiochem (Läu-
felfingen, Switzerland), Bachem (Bubendorf, Switzerland), Iris Bio-
tech (Marktredwitz, Germany), Aldrich (Milwaukee, WI), Acros
(Geel, Belgium), Neosystem (Strasbourg, France), and Luxem-
bourg Industries (Tel Aviv, Israel). Analytical HPLC was carried
out with a Waters instrument, comprising two solvent-delivery
pumps and automatic injector (Waters Separations Module 2695)
and a variable-wavelength detector (model Waters 996 Photodiode
Array). UV detection was performed at 220 nm, and linear gradi-
ents of CH3CN (0.036% TFA) into H2O (0.045% TFA) were run
at a flow rate of 1.0 mL/min. MS-HPLC was carried out with a
Waters instrument, comprising two solvent-delivery pumps and
automatic injector (Waters Separations Module 2795), a dual-wave-
length detector (Waters 2487, Dual λ Absorbance Detector), and
an electrospray detector (Waters micromass ZQ). NMR spectra
were acquired with a Mercury-400 (400 MHz) spectrometer (High
Field NMR Unit, Barcelona Science Park), data are given on the
pNZ-D
-Val-OH: M.p. 125.5–127.5 °C.[8a] HPLC: Rt = 10.5 min
(Gradient A). IR (KBr): ν = 3322, 2970, 1693, 1540, 1351 cm–1. 1H
˜
NMR (400 MHz, DMSO): δ = 8.22 (d, J = 8.6 Hz, 2 H), 7.63 (d,
J = unresolved, NH), 7.61 (d, J = 8.6 Hz, 2 H), 5.17 (s, 2 H), 3.86
(dd, J = 8.5, 5.9 Hz, 1 H), 2.48 (m, 1 H), 0.89 (d, J unresolved, 3
H), 0,87 (d, J unresolved, 3 H) ppm. 13C NMR (100 MHz,
DMSO): δ = 173.82 (C), 156.81 (C), 147.61 (C), 145,75 (C), 128.77
(CH Ar), 124.18 (CH Ar), 64.91 (CH2), 60.30 (CH), 30.23 (CH),
19.83 (CH3), 18.68 (CH3) ppm. HRMS (MALDI-TOF): m/z calcd.
for C13H16N2O6Na [M + Na+] 319.0906, found 319.0889.
pNZ-Gly-OH: M.p. 121.5–122.5 °C (ref.[8a] 122.5–124 °C). HPLC:
R = 4.50 min (Gradient B). IR (KBr): ν = 3383, 3117, 2941, 1751,
˜
t
1705, 1522, 1350 cm–1. H NMR (400 MHz, DMSO): δ = 8.21 (d,
1
J = 8.7 Hz, 2 H), 7.70 (t, J = 6.1 Hz, NH), 7.59 (d, J = 8.7 Hz, 2
H), 5.18 (s, 2 H), 3.67 (d, J = 6.1 Hz, 2 H) ppm. 13C NMR
(100 MHz, DMSO): δ = 172.12 (C), 156.9 (C), 147.62 (C), 145.7
(C), 128.77 (CH), 124.20 (CH), 64.95 (CH2), 42.84 (CH2) ppm.
HRMS (CI): m/z calcd. for C10H11N2O6 [M + H+] 255.0618, found
255.0611.
1
δ scale referenced to TMS (see Supporting Information for H and
pNZ-Leu-OH: M.p. 81.0–85.5 °C (ref.[8a] 60–61 °C). HPLC: Rt =
13C NMR spectra). Amino acid analyses were performed using a
Beckman System 6300 High Performance Analyzer. Resins were
treated with a mixture of HCl and propionic acid (1:1) at 160 °C
for 1 h, and after evaporating the acid under reduced pressure, they
were suspended in amino acid analysis buffer and filtered.
7.90 min (Gradient B). IR (KBr): ν = 3421.3, 2961, 1693, 1524,
˜
1
1345 cm–1. H NMR (400 MHz, DMSO): δ = 8.22 (d, J = 8.8 Hz,
2 H), 7.70 (d, J = 8.2 Hz, NH), 7.59 (d, J = 8.8 Hz, 2 H), 5.16 (d,
J = 1.96 Hz, 2 H), 3.95 (m, 1 H), 1.63 (m, 1 H), 1.50 (m, 2 H),
0.87 (d, J = 6.6 Hz, 3 H), 0.83 (d, J = 6.5 Hz, 3 H) ppm. 13C NMR
(100 MHz, DMSO): δ = 174.93 (C), 156.55 (C), 147.61 (C), 145.73
(C), 128.75 (CH), 124.19(CH), 64.87 (CH2), 52.94 (CH), 40.27
(CH2), 25.00 (CH), 23.54 (CH3), 21.80 (CH3) ppm. HRMS (CI):
m/z calcd. for C14H19N2O6 [M + H+] 311.1244, found 311.1252.
pNZ-Amino Acid Synthesis
Method 1: p-Nitrobenzyl chloroformate (1.73 g, 8 mmol) was dis-
solved in 1,4-dioxane (3.5 mL) and a solution of sodium azide
(0.624 g, 9.6 mmol) in H2O (2.5 mL) was added. The resulting
emulsion was stirred for 2 h and the formation of the azide was
monitored by TLC (CH2Cl2). Gly (0.600 g, 8 mmol), dissolved in
1,4-dioxane/2% aqueous Na2CO3 (1:1) (10 mL), was then added
dropwise, and the resulting white suspension was stirred for 24 h,
keeping the pH between 9 and 10 by adding 10% aqueous Na2CO3.
At this point, TLC (CH2Cl2) showed that there was no remaining
azide, H2O (75 mL) was added and the suspension was washed with
methyl tert-butyl ether (MTBE) (3×40 mL). The aqueous portion
was acidified to pH = 2–3 with 12 n HCl/H2O (3:1) and a white
precipitate appeared, which was filtered off and dried to yield
1.44 g (71%) of the title compound as a white solid.
pNZ-Ile-OH: M.p. 79–83 °C (ref.[8a] 77.5–80 °C). HPLC: Rt
=
11.21 min (Gradient A). IR (KBr): ν = 3321, 2967, 1727, 1659,
˜
1538, 1349 cm–1. 1H NMR (400 MHz, DMSO): δ = 8.22 (d, J =
8.7 Hz, 2 H), 7.64 (d, J = 8.5 Hz, NH), 7.61 (d, J = 8.7 Hz, 2 H),
5.17 (s, 2 H), 3.90 (dd, J = 8.5, 6.1 Hz, 1 H), 2.48 (m, 1 H), 1.77
(m, 1 H), 1.38 (m, 1 H), 1.20 (m, 1 H), 0.86 (d, J = 6.8 Hz, 3 H),
0.82 (t, J = 7.4 Hz, 3 H) ppm. 13C NMR (100 MHz, DMSO): δ =
173.84 (C), 156.72 (C), 147.62 (C), 145.76 (C), 128.78 (CH), 124.19
(CH), 64.92 (CH2), 59.32 (CH), 36.76 (CH), 25.35 (CH2), 16.29
(CH3), 11.95 (CH3) ppm. HRMS (MALDI-TOF): m/z calcd. for
C14H18N2O6Na [M + Na+] 333.1063 found 333.1063.
Method 2: The synthesis was performed as in Method 1 but the
acidic suspension was extracted with EtOAc. The organic layers
were dried with MgSO4, filtered, and the solvent was removed un-
der reduced pressure to yield an oil, which solidified as white solid
by washing with diethyl ether or suspended in acetonitrile (AcCN)/
H2O and lyophilized (H-d-Val-OH and H-l-Ala-OH). The pNZ-
amino acids were recrystalized in H2O or in diethyl ether/hexane.
pNZ-Tyr(tBu)-OH: M.p. 130–136.5 °C. HPLC: Rt = 9.61 min
(Gradient B). IR (KBr): ν = 3351, 2980, 1703, 1522, 1352 cm–1. 1H
˜
NMR (400 MHz, DMSO): δ = 8.18 (d, J = 8.7 Hz, 2 H), 7.69 (d,
J = 8.6 Hz, NH), 7.45 (d, J = 8.7 Hz, 2 H), 7.13 (d, J = 8.4 Hz, 2
H), 6.84 (J = 8.4 Hz, 2 H), 5.09 (dd, J = 24,1 Hz, 14,2 Hz, 2 H),
4.15 (m, 1 H), 3.04 (dd, Jgem = 13.8, J = 4.2 Hz, 1 H), 2.77 (dd,
Jgem = 13.8, J = 10.7 Hz, 1 H), 1.24 (s, 9 H) ppm. 13C NMR
(100 MHz, DMSO): δ = 173.99 (C), 156.28 (C), 154.15 (C), 147.51
(C), 145.86 (C), 133.22 (C), 130.35 (CH), 128.46 (CH), 124.11
(CH), 123.95 (CH), 78.27 (C), 64.62 (CH2), 56.47 (CH), 36.70
(CH2), 29.21 (CH3) ppm. HRMS (CI): m/z calcd. for C21H25N2O7
[M + H+] 417.1663, found 417.1665.
All protected amino acids were characterized by HPLC [Gradient
A: from H2O to AcCN; Gradient B: from AcCN/H2O (3:7) to
AcCN, where H2O contained 0.045% of TFA and AcCN contained
1
0.036% of TFA], HPLC-MS, H and 13C NMR, HR-MS, and IR;
for used method and yields see Table 3.
3036
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2005, 3031–3039

pNZ as Nα-Protecting Group in Orthogonal Solid-Phase Peptide Synthesis
FULL PAPER
Table 3. Method used and yield for the preparation of pNZ-amino acids.
Amino acid
Leu
Ile
D-Val
Phe
Gly
Ser(tBu) Orn(Boc) Asp(OtBu)
Ala
Tyr(tBu)
Method
Yield [%]
2
77
2
94
2
83
1
84
1
71
2
85
2
83
2
89
2
81
1
82
pNZ-Phe-OH: M.p. 129.0–135.1 °C (ref.[8b] 134.5–136.5 °C). reagent [DIPCDI (4 equiv.) and HOBt (4 equiv.), or HATU
HPLC: R = 7.90 min (Gradient B). IR (KBr): ν = 3329, 1702, (3.8 equiv.) and DIEA (12 equiv.)] in DMF (0.5 mL) to the resin
˜
t
1516, 1346 cm–1. 1H NMR (400 MHz, DMSO): δ = 8.17 (d, J =
8.7 Hz, 2 H), 7.76 (d, J = 8.5 Hz, NH), 7.48 (d, J = 8.7 Hz, 2 H),
7.25 (m, 5 H), 5.10 (s, 2 H), 4.16 (m, 1 H), 3.08 (dd, Jgem = 13.8,
J = 4.3 Hz, 1 H), 2.82 (dd, Jgem = 13.8, J = 10.6 Hz, 1 H) ppm.
13C NMR (100 MHz, DMSO): δ = 173.88 (C), 156.34 (C), 147.53
(C), 145.76 (C), 138.61 (C), 129.80 (CH), 128.83 (CH), 128.57
(CH), 127.01 (CH), 124.12 (CH), 64.74 (CH2), 56.33 (CH), 37.20
(CH2) ppm. HRMS (CI): m/z calcd. for C17H17N2O6 [M + H+]
345.1087, found 345.1079.
and stirring the mixture for 60–90 min. For all cases, reaction pro-
gress was monitored by the ninhydrin test[39] and couplings were
repeated if a positive test result was obtained. Peptide synthesis
transformations and washes were performed at 25 °C unless other-
wise indicated.
Final Cleavage and Deprotection: The acidolytic cleavage was car-
ried out with TFA/H2O/DCM (90:5:5) (10 mL/g of resin). The TFA
was removed by evaporation and the residue was taken up in
HOAc/H2O (7:3), washed three times with CHCl3, and the aqueous
phase lyophilized.
pNZ-Ala-OH: Amorphous solid (ref.[8b] 132.5–134 °C). HPLC: Rt
= 9.28 min (Gradient A). IR (KBr): ν = 3348, 1697, 1530,
˜
Synthesis of H-Tyr-Phe-Gly-Gly-Leu-NH2
1
1351 cm–1. H NMR (400 MHz, DMSO): δ = 8.21 (d, J = 8.7 Hz,
Synthesis 1: Rink amide resin (50 mg, 0.66 mmol/g). Couplings
DIPCDI/HOBt method. Then, the pNZ was removed and the resin
was neutralized with DIEA/CH2Cl2 (1:9), washed with DCM
(5×1 min), and DMF (5×1 min) before performing the next coup-
ling. After final acydolitic cleavage and workup, the crude product
was analyzed by HPLC [Rt = 6.37 min; gradient: from H2O
(0.045% of TFA) to AcCN (0.036% of TFA)], and HPLC-MS.
2 H), 7.74 (d, J = 7.6 Hz, NH), 7.59 (d, J = 8.7 Hz, 2 H), 5.16 (s,
2 H), 3.99 (m, 1 H), 1.26 (d, J = 7.4 Hz, 3 H) ppm. 13C NMR
(100 MHz, DMSO): δ = 174.94 (C), 156.23 (C), 147.61 (C), 145.71
(C), 128.79 (CH), 124.18 (CH), 64.84 (CH2), 49.97 (CH), 17.70
(CH3) ppm. HRMS (MALDI-TOF): m/z calcd. for C11H12N2O6Na
[M + Na+] 291.0593, found 291.0599.
pNZ-Orn(Boc)-OH: Amorphous solid. HPLC: Rt = 7.51 min (Gra-
dient B). IR (KBr): ν = 3405, 2978, 1717, 1523, 1348 cm–1. 1H
Synthesis 2: The same synthetic process as outline above was re-
˜
NMR (400 MHz, DMSO): δ = 8.22 (d, J = 8.6 Hz, 2 H), 7.68 (d,
J = 8.0 Hz, NH), 7.60 (d, J = 8.6 Hz, 2 H), 6.77 (t, J = 4.8 Hz,
NH), 5.17 (s, 2 H), 3.90 (m, 1 H), 2.91 (m, 2 H), 1.58 (m, 1 H),
1.53 (m, 1 H), 1.42 (m, 2 H), 1.35 (s, 9 H) ppm. 13C NMR
(100 MHz, DMSO): δ = 174.40 (C), 156.50 (C), 147.60 (C), 145.77
(C), 128.73 (CH), 124.19 (CH), 78.07 (C), 64.84 (CH2), 54.45 (CH),
40.09 (CH2), 28.94 (CH3), 28.88 (CH2), 26.87 (CH2) ppm. HRMS
(CI): m/z calcd. for C18H26N3O8 [M + H+] 412.1721, found
412.1719.
peated but carrying out the removal of the pNZ at 50 °C.
Synthesis of H-Ala-Leu-Ser-Tyr-Gly- Phe-NH2: Rink amide resin
(50 mg, 0.66 mmol/g). Couplings: pNZ-amino acids (4 equiv.),
HATU (3.8 equiv.) and DIEA (12 equiv.) in DMF (0.5 mL) were
added to the resin and the reaction mixture was stirred for 1 h.
Then, the pNZ group was removed and the resin was washed before
performing the next coupling without previous neutralization. Af-
ter final acydolitic cleavage and workup, the crude product was
analyzed by HPLC [Rt = 6.05 min; gradient: from H2O/AcCN
(95:5) to H2O/AcCN (5:95) where H2O contained 0.045% of TFA
and AcCN contained 0.036% of TFA] and HPLC-MS.
pNZ-Asp(OtBu)-OH: M.p. 79.5–82.6 °C. HPLC: Rt = 11.37 min
(Gradient A). IR (KBr): ν = 3342, 3157, 1723, 1699, 1521,
˜
1
1349 cm–1. H NMR (400 MHz, DMSO): δ = 8.21 (d, J = 8.7 Hz,
Tests of DKP Formation: Aminomethyl resin (100 mg,
f =
2 H), 7.76 (d, J = 8.6 Hz, NH), 7.59 (d, J = 8.7 Hz, 2 H), 5.17 (s,
2 H), 4.33 (m, 1 H), 2.68 (dd, Jgem = 16.0, J = 5.7 Hz, 1 H), 2.52
(dd, Jgem = 16.0, J = 8.4 Hz, 1 H), 1.35 (s, 9 H) ppm. 13C NMR
(100 MHz, DMSO): δ = 173.03 (C), 169.87 (C), 156.21 (C), 147.62
(C), 145.62 (C), 128.82 (CH), 124.16 (CH), 80.98 (C), 64.97 (CH2),
51.32 (CH), 37.88 (CH2), 28.31 (CH3) ppm. HRMS (CI): m/z calcd.
for C16H21N2O8 [M + H+] 369.1299, found 369.1309.
1.2 mmol/g) was used as the base resin. Fmoc-l-Pro-OH (4 equiv.),
DMAP (0.4 equiv.) and DIPCDI (4 equiv.) in DMF (1 mL) were
added to the HO-AB-Leu-aminomethyl resin, which is a Wang-
type resin,[40] and the reaction mixture was stirred for 1 h. After
washing, the TosCl-PNBP test[41] confirmed the correct coupling
of the amino acid. Then the Fmoc group was removed, the resin
was divided into two parts and the peptides were synthesized.
Solid-Phase Synthesis: Solid-phase syntheses were carried out in
polypropylene syringes (2–10 mL) fitted with a porous polyethylene
disk. Solvents and soluble reagents were removed by suction.
Fmoc Synthesis: The Fmoc-d-Val-OH was incorporated using
DIPCDI/HOBt-mediated coupling, the Fmoc was removed; after
washings, the Fmoc-Phe-OH was incorporated using the same pro-
tocol, the Fmoc was removed and the amino acid hydrolysis of a
peptide resin aliquot indicated that the extension of side reactions
has been of 100%.
Fmoc: Deprotection was accomplished with piperidine/DMF (2:8)
(2×3 min, 1×4 min, and 1×5 min). Washings between deprotec-
tion, coupling, and final deprotecion steps were carried out in
DMF (5×1 min) and DCM (5×1 min) using 10 mL solvent/g resin
for each wash.
pNZ Synthesis: pNZ-d-Val-OH was incorporated using DIPCDI/
HOBt-mediated coupling, the pNZ was removed; after washings,
the Fmoc-Phe-OH (4 equiv.), PyAOP (4 equiv.),[42] and DIEA
(8 equiv.) in DMF (0.5 mL) were added and the reaction mixture
was stirred for 1 h. After washing as indicated above, the ninhydrin
test was negative. The resin was then washed and the amino acid
hydrolysis of a peptide resin aliquot indicated that side reactions
did not occur.
pNZ: Unless otherwise indicated, deprotection was accomplished
with 6 m SnCl2 and 1.6 mm HCl/dioxane, in DMF (2×30 min). The
resin was then washed with DMF (3×30 s), DMF/H2O (3×30 s),
THF/H2O (3×30 s), DMF (3×30 s), and DCM (3×30 s).
Coupling of Fmoc-Amino Acids and Handles: This was performed
by adding the carboxylic acid reagent (4 equiv.) and the coupling
Eur. J. Org. Chem. 2005, 3031–3039
www.eurjoc.org
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3037

A. Isidro-Llobet, J. Guasch-Camell, M. Álvarez, F. Albericio
FULL PAPER
[13]
[14]
For a broad discussion of this subject, see: L. J. Cruz, N. G.
Beteta, A. Ewenson, F. Albericio, Org. Process Res. Dev. 2004,
8, 920.
Tests of Aspartimide Formation: Rink amide resin (200 mg,
0.66 mmol/g) was used and the synthesis was carried out as out-
lined above using DIPCDI/HOBt-mediated couplings. After re-
moval of the Fmoc group of the Gly, the resin was divided into
two parts for the Fmoc and the pNZ synthesis. After acidolytic
cleavage and workup, the crude products were analyzed by HPLC
[Rt = 9.04 min; gradient: from H2O to H2O/AcCN (7:3), where
H2O contained 0.045% of TFA and AcCN contained 0.036% of
TFA] and HPLC-MS.
Although the existence of this side reaction was not mentioned,
Fmoc-N3 was already proposed as an alternative to the chloro-
formate in the seminal papers of Carpino and Han: L. A. Car-
pino, G. Y. Han, J. Am. Chem. Soc. 1970, 92, 5748 and L. A.
Carpino, G. Y. Han, J. Org. Chem. 1972, 37, 3404.
In the literature, the p-azidobenzyloxycarbonyl group, which
behaves similarly to pNZ, is also described. The reduction of
the azide with dithiothreitol (DTT) or SnCl2 should lead to the
p-aminobenzyloxycarbonyl derivative, which liberates the free
amine by the same mechanism as pNZ. However, in our hands
this azide derivative gave substantially worse results than pNZ.
a) B. Loubinoux, P. Gerardin, Tetrahedron Lett. 1991, 32, 351;
b) R. J. Griffin, E. Evers, R. Davison, A. E. Gibson, D. Layton,
W. J. Irwin, J. Chem. Soc., Perkin Trans. 1 1996, 1205.
J.-F. Wen, W. Hong, K. Yuan, T. C. W. Mak, H. N. C. Wong,
J. Org. Chem. 2003, 68, 8918, and references therein.
J. T. Manka, F. Guo, J. Huang, H. Yin, J. M. Farrar, M. Sien-
kowska, V. Benin, P. Kaszynski, J. Org. Chem. 2003, 68, 9574,
and references therein.
[15]
Acknowledgments
This work was partially supported by CICYT (BQU, 2003-00089),
the Generalitat de Catalunya (Grup Consolidat and Centre de Re-
ferència en Biotecnologia), and the Barcelona Science Park. A. I.
thanks MECD (Spain) for a predoctoral fellowship.
[16]
[17]
[1] a) R. B. Merrifield, J. Am. Chem. Soc. 1963, 85, 2149; b) R. B.
Merrifield, Angew. Chem. Int. Ed. Engl. 1985, 24, 799; c) R. B.
Merrifield, Science 1986, 232, 341.
[2] S. Lien, H. B. Lowman, Trends Biotechnol. 2003, 21, 556.
[3] a) A. Loffet, J. Pept. Sci. J. Peptide Sci. 2002, 8, 1; b) T. Bruck-
dorfer, O. Marder, F. Albericio, Current Pharm. Biotech. 2004,
5, 29.
[4] a) P. Lloyd-Williams; F. Albericio; E. Giralt, Chemical Ap-
proaches to the Synthesis of Peptides and Proteins, CRC, Boca
Raton, FL, USA, 1997; b) T. S. Yokum, G. Barany, in: Solid-
phase Synthesis. A Practical Guide, Marcel Dekker Inc., New
York, USA, 2000, pp. 79–102; c) G. B. Fields, J. L. Lauer-Fi-
elds, R.-q. Liu, G. Barany, in: Synthetic Peptides: A User’s
Guide (Eds.: G. A. Grant), 2nd ed., W. H. Freeman & Co., New
York, 2001, pp. 93–219; d) M. Goodman, A. Felix, L. A. Mor-
oder, C. Toniolo (Eds.), Houben-Weyl, vol. E22a–e (“Synthesis
of Peptides and Peptidomimetics”), Georg Thieme Verlag,
Stuttgart, Germany, 2002.
[18]
[19]
a) W. Liao, C. F. Piskorz, R. D. Locke, K. L. Matta, Bioorg.
Med. Chem. Lett. 2000, 10, 793; b) X. Qian, O. Hindsgaul,
Chem. Commun. 1997, 1059, and references therein.
a) A. A. Pletnev, Q. Tian, R. C. Larock, J. Org. Chem. 2002;
67, 9276; b) M. Schlitzer, M. Böhm, I. Sattler, H.-M. Dahse,
Bioorg. Med. Chem. 2000, 8, 1991.
[20]
[21]
SnCl2 has also been used to remove the pNB ester on solid
phase, see ref.[10]
a) E. Guibé-Jampel, M. Wakselman, Synth. Commun. 1982, 12,
219; b) R. A. Scheurerman, D. Tumelty, Tetrahedron Lett.
2000, 41, 6531.
If aminium/uronium salt coupling methods are used, this neu-
tralization step can be omitted. See synthesis of phospholipase
A2 (18–23).
B. F. Gisin, R. B. Merrifield, J. Am. Chem. Soc. 1972, 94, 3102.
M. C. Khosla, R. R. Smeby, F. M. Bumpus, J. Am. Chem. Soc.
1972, 94, 3102.
For instance, the peptide-resin anchorage: DKP formations are
favored when the peptide is bound to the resin as a benzyl
ester; the nature of the first and second amino acids of the
peptide: the presence of Pro, Gly or of N-alkylamino acids can
lead to a substantial extent of DKP formation because all of
them favor the cis configuration of the amide bond, which is
the optimal configuration to form DKPs. Another unfavorable
combination is to have one d- and one l-amino acid in the
dipeptide because it decreases the steric hindrance during the
cyclization. See ref.[4a]
E. Pedroso, A. Grandas, X. de las Heras, R. Eritja, E. Giralt,
Tetrahedron Lett. 1986, 27, 743.
a) K. Suzuki, K. Nitta, N. Endo, Chem. Pharm. Bull. 1975, 23,
222; b) M. Gairi, P. Lloyd-Williams, F. Albericio, E. Giralt,
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a) K. Akaji, Y. Kiso, L. A. Carpino, J. Chem. Soc., Chem.
Commun. 1990, 584; b) K. Barlos, D. Gatos, W. Schaefer, An-
gew. Chem. Int. Ed. Engl. 1991, 30, 590; c) C. Chiva, M. Vila-
seca, E. Giralt, F. Albericio, J. Pept. Sci. 1999, 5, 131.
J. Alsina, E. Giralt, F. Albericio, Tetrahedron Lett. 1996, 37,
4195.
N. Thieriet, J. Alsina, E. Giralt, F. Guibé, F. Albericio, Tetrahe-
dron Lett. 1997, 38, 7275.
The sequence d-Val-Pro is prone for DKP formation because
it contains the combination of one d- and one l-amino acid
with a Pro residue. On the other hand, the Val is a β-branched
residue and therefore its acylation is not trivial. See
refs.[4a,26,27b,29]
[22]
[23]
[24]
[25]
[5] W. C. Chan; P. D. White (Eds.), Fmoc Solid Phase Peptide Syn-
thesis, Oxford University Press, Oxford, UK, 2000.
[6] The orthogonal concept is based on the use of independent
classes of protecting groups, removed by different mechanisms
so that they may be removed in any order and in the presence
of all other types of groups. a) G. Barany, R. B. Merrifield, J.
Am. Chem. Soc. 1977, 99, 7363; b) G. Barany, F. Albericio, J.
Am. Chem. Soc. 1985, 107, 4936.
[7] F. Albericio, Biopolymers 2000, 55, 123.
[8] a) F. H. Carpenter, D. T. Gish, J. Am. Chem. Soc. 1952, 74,
3818; b) D. T. Gish, F. H. Carpenter, J. Am. Chem. Soc. 1953,
75, 950.
[26]
[27]
[9] a) J. E. Shields, F. H. Carpenter, J. Am. Chem. Soc. 1961, 83,
3066; b) M. D. Hocker, C. G. Caldwell, R. W. Macsata, M. H.
Lyttle, Pept. Res. 1995, 8, 310; c) S. Peluso, P. Dumy, C. Nkub-
ana, Y. Yokokawa, M. Mutter, J. Org. Chem. 1999, 64, 7114.
[10] a) P. Romanovskis, A. F. Spatola, J. Pept. Res. 1998, 52, 356;
b) M. Royo, J. Farrera-Sinfreu, L. Solé, F. Albericio, Tetrahe-
dron Lett. 2002, 43, 2029.
[11] a) M. D. Bachi, J. Ross-Peterson, J. Chem. Soc., Chem. Com-
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For couplings without preactivation of the protected amino
acid, phosphonium salts such as PyAOP are preferred to amin-
ium/uronium salts, because the latter can give guanidinium for-
3038
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Eur. J. Org. Chem. 2005, 3031–3039

pNZ as Nα-Protecting Group in Orthogonal Solid-Phase Peptide Synthesis
FULL PAPER
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[42] (Benzotriazol-1-yloxy)tris(pyrrolidino)phosphonium hexafluo-
rophosphate (PyBOP) can also be used instead of PyAOP.
Received: March 6, 2005
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© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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