Oxime carbonates: Novel reagents for the introduction of fmoc and alloc protecting groups, free of side reactions

Article abstract of DOI:10.1002/ejoc.201000028

Fmoc and Alloc protecting groups represent a consistent alternative to classical Boc protection in peptide chemistry. The former was established in the last decades as the α-amino protecting group of choice, whereas the latter allows a fully orthogonal protection strategy with Fmoc and Boc. Usually, the introduction of the Fmoc and Alloc moieties takes place through their halogenoformates, azides, or activated carbonates. This rather simple reaction is accompanied by several side reactions, specially the formation of Fmoc/Alloc dipeptides and even tripeptides. The present work describes new promising Fmoc/Alloc-oxime reagents, which are easy to prepare, stable, and highly reactive crystalline materials that afford almost: contaminant-free Fmoc/Alloc-amino acids in high yields by following a conventional procedure. Amongst the Fmoc-oxime derivatives, the N-hydroxypicolimmidoyl cyanide derivative (N-([(9H-fluoren-9-yl)methoxy]carbonyloxy}picolinimidoyl cyanide) gave the best results for the preparation of Fmoc-Gly-OH, which is the most predisposed to give side reactions. The same Alloc-oxime analogue afforded the preparation of Alloc-Gly-OH in good yield, purity, and extremely low dipeptide formation, as analyzed by reverse-phase HPLC and NMR spectroscopy.

Full text of DOI:10.1002/ejoc.201000028

FULL PAPER
DOI: 10.1002/ejoc.201000028
Oxime Carbonates: Novel Reagents for the Introduction of Fmoc and Alloc
Protecting Groups, Free of Side Reactions
Sherine N. Khattab, Ramon Subirós-Funosas,[ bb , ,cc ],eA] yman El-Faham,*[a,b,d] and
[a]
Fernando Albericio*[
Keywords: Amino acids / Protecting groups / Peptides / Oximes
Fmoc and Alloc protecting groups represent a consistent al-
ternative to classical Boc protection in peptide chemistry. The
former was established in the last decades as the α-amino
protecting group of choice, whereas the latter allows a fully
orthogonal protection strategy with Fmoc and Boc. Usually,
the introduction of the Fmoc and Alloc moieties takes place
prepare, stable, and highly reactive crystalline materials that
afford almost contaminant-free Fmoc/Alloc-amino acids in
high yields by following a conventional procedure. Amongst
the Fmoc-oxime derivatives, the N-hydroxypicolinimidoyl
cyanide derivative (N-{[(9H-fluoren-9-yl)methoxy]carbonyl-
oxy}picolinimidoyl cyanide) gave the best results for the
through their halogenoformates, azides, or activated carbon- preparation of Fmoc-Gly-OH, which is the most predisposed
ates. This rather simple reaction is accompanied by several
side reactions, specially the formation of Fmoc/Alloc dipep-
tides and even tripeptides. The present work describes new
promising Fmoc/Alloc-oxime reagents, which are easy to
to give side reactions. The same Alloc-oxime analogue af-
forded the preparation of Alloc-Gly-OH in good yield, purity,
and extremely low dipeptide formation, as analyzed by re-
verse-phase HPLC and NMR spectroscopy.
Introduction
general basic and acidic conditions, it introduces orthogo-
nality towards Boc and Fmoc, being of special interest as
side-chain protection in the synthesis of cyclic peptides,
when the so-called three dimensional protection strategy is
An appropriate choice of the protection strategy is essen-
tial to the success of peptide[ synthesis, conditioning the
yield and purity of the desired product. To meet that pur-
pose, several protecting groups have been proposed that of-
1]
[8]
used. Its removal can be easily and selectively achieved
with tetrakis(triphenylphosphane)palladium(0) and a suit-
fer a wide range of removal conditions, enabling the re-
[9,10]
able nucleophile/scavenger.
quired orthogonality.[
2,3]
9-Fluorenylmethyloxycarbonyl
The use of chloroformates represents the most potent
amino acids (Fmoc-amino acids) are potentially amongst
the most versatile intermediates for peptide synthesis, espe-
cially when used in conjunction with acid-labile side-chain
protecting groups, being considered as a milder alternative
strategy for the N-protection with Fmoc and Alloc moie-
[
11]
ties. Nevertheless, it has been reported that the high reac-
tivity of these chlorides, such as Fmoc-Cl (1) or Alloc-Cl
(2), might lead to the formation of amino-acid-based by-
[4,5]
to the more classical Boc strategy.
Alloc) group has also been proposed for the preparation
The allyloxycarbonyl
products, including the protection of bulky residues, that
(
[
12–16]
become inserted into the peptide chain.
As result of
of N-urethane blocked amino acids, successfully allowing
the synthesis of antitumoral peptides when used as tempo-
rary α-amino protecting group.[6,7] Due to its stability to
this detrimental side reaction, considerable formation of
Fmoc/Alloc-dipeptides and tripeptides (1–20%) has been
observed, as established by HPLC, amino acid analysis,
NMR spectroscopy, and the synthesis of a number of stan-
[
a] Department of Chemistry, Faculty of Science, Alexandria Uni-
versity,
[
13]
dards. A previous “in situ” bis-trimethylsilylation step of
protection of the residue, before conducting the reaction
with the chloroformate, can prevent the formation of such
Ibrahimia 21321, P. O. Box 246, Alexandria, Egypt
Fax: +203-5841866
E-mail: aymanel_faham@hotmail.com
[
15,17]
[
b] Institute for Research in Biomedicine, Barcelona Science Park, byproducts.
In fact, Fmoc/Alloc-oligopeptides are
Baldiri Reixac 10, 08028 Barcelona, Spain
likely formed through the intermediary formation of rela-
tively stable mixed anhydrides when highly reactive chloro-
Fax: +34-93-403-71-26
E-mail: albericio@irbbarcelona.org
[
18,19]
[
[
[
c] CIBER-BBN, Networking Centre on Bioengineering, Biomate- formates are used (Scheme 1, path B).
rials and Nanomedicine, Barcelona Science Park,
The drawbacks associated to these powerful reagents
d] King Saud University, College of Science, Department of prompted the evaluation of less reactive approaches for
Baldiri Reixac 10, 08028 Barcelona, Spain
Chemistry,
the introduction of N-blocking groups, like the 1,2,2,2-
Riyadh 11451, P. O. Box 2455, Kingdom of Saudi Arabia
[
20,21]
[22]
tetrachloroethyl,
pentafluorophenyl,
5-norbornene-2,3-dicarboximido,
symmetrical pyrocarbonates,
e] University of Barcelona, Department of Organic Chemistry,
Martí i Franqués 1-11, 08028 Barcelona, Spain
[
23]
[24]
and
Eur. J. Org. Chem. 2010, 3275–3280
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3275

S. N. Khattab, R. Subirós-Funosas, A. El-Faham, F. Albericio
FULL PAPER
Scheme 1. Side reaction during Fmoc protection.
Scheme 2. Synthesis of Fmoc-oxime and N-hydroxypyridinone de-
-MBt (2-mercaptobenzothiazole),[25] although only the hy- rivatives.
droxysuccinimido (Su) carbonate found general accept-
2
[
13–16,26,27]
ance.
Nonetheless, the formation of up to 0.4%
The Fmoc-introducing reagents were afforded by this
method in high yields (Ͼ80% in all analogues synthesized)
and purity, according to the found elemental analysis, com-
pared to the calculated values (Table 1). Fmoc-oximes 3–6
and Fmoc-hydroxypyridinone 7 were obtained as white sol-
ids after recrystallization in CH Cl /hexane, except for di-
ethylcarboxylate analogue 3, which was an oil at room tem-
perature, and therefore, its handling was tedious in com-
parison to the rest of the derivatives.
of some amino-acid-based byproducts like Fmoc/Alloc-β-
Ala-OH and Fmoc/Alloc-β-Ala-AA-OH can occur when
using this reagent for α-amino protection.[ A detailed dis-
cussion of the mechanism of this side reaction has been
proposed by Isidro-Llobet et al.[ Azide derivatives, which
can be used as solids after isolation from the chloro-
28]
25]
2
2
[
12,29]
formates
amino acid,
or formed in situ before reacting with the
have also been proposed as an alternative
[
30]
pathway to the N-protection of amino acids, although its
explosive nature compromises its use in large-scale synthe-
sis.
Table 1. Yield, m.p., and elemental analysis of the protecting
groups.
Our group has recently been interested in the search of a
new family of N-hydroxy-containing compounds as replace-
ments for 1-hydroxybenzotriazole (HOBt) and its deriva-
tives. Some ketoximes, bearing various electron-with-
drawing substituents were considered for that purpose, as
they are rather stable compared to oximes with hydrogen
Yield
%]
M.p.
Elemental analysis calculated (found)
[
[°C]
–[a]
174–175
150–151
165–166
195–196
C
H
N
3
4
5
6
7
84
91
87
93
89
64.23 (64.44) 5.14 (5.30)
65.93 (66.14) 4.43 (4.70)
3.40 (3.63)
7.69 (7.91)
68.14 (68.39) 3.49 (3.61) 13.24 (13.48)
71.54 (71.77) 4.09 (4.32) 11.38 (11.53)
[31]
atoms in the α-carbon position, and they also contain a
highly acidic N-hydroxy moiety, which results in an excel-
lent leaving group. A deep study on ethyl 2-cyano-2-hy-
droxyiminoacetate (Oxyma) was undertaken, showing a
high reduction of racemization and coupling efficiency in
72.06 (72.31) 4.54 (4.68)
4.20 (4.41)
[
a] Ca. 20 °C; oil at room temperature.
[
32–35]
Due to its size, H-Gly-OH is one of the amino acids that
hindered sequences when this additive was employed.
becomes most contaminated during its α-amino protection.
When Fmoc-Gly-OH was synthesized by using Fmoc-Cl (1)
in dioxane/aqueous sodium carbonate following the stan-
dard procedure described in the literature, the product was
contaminated with a major (10–20%) amount of Fmoc-
Therefore, this oxime can be converted into promising car-
bonate-type reagents for the introduction of Fmoc and Al-
loc.
[
13,18,19]
Gly-Gly-OH, as found by TLC.
residue has been chosen as a model for carrying out a care-
For this reason, this
Results and Discussion
The suitability of these oximes as leaving groups in car- ful in-depth study. The reaction was carried out overnight
bonate-type reagents for Fmoc/Alloc introduction was first in water/dioxane, in the presence of sodium hydrogen car-
[
37]
tested in the synthesis and evaluation of Fmoc derivatives. bonate.
In general, Fmoc-oxime reagents were quite
Therefore, preparation of Fmoc-oximes 3–6 and (9H-flu- stable to competitive hydrolysis by NaOH, Na CO , or tri-
2
3
oren-9-yl)methyl-2-oxopyridin-1(2H)-yl carbonate (7) was ethylamine in the solvent mixture at room temperature.
readily achieved by reaction of the corresponding oximes Compound 3 (diethylcarboxylate derivative) dissolves only
or N-hydroxy-2-pyridinone (HOPO)[ with Fmoc-Cl (1) in partly in a solvent water mixture at an initial stage of the
36]
the presence of sodium carbonate, following a well-reported reaction, but the mixture usually becomes homogeneous
method described for other reagents (Scheme 2).[
26]
within 1 h, whereas in the case of 6 (cyanopyridyl deriva-
3276
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Eur. J. Org. Chem. 2010, 3275–3280

Novel Reagents for the Introduction of Fmoc and Alloc Protecting Groups
tive) the reaction becomes homogeneous after 10–15 min. garding Alloc. The formation of the Alloc-cyanopyridyl-
The results obtained in the preparation of Fmoc-Gly-OH oxime reagent was accomplished by using the potassium
with reagents 3–7 are summarized in Table 2.
salt of the oxime 8, previously isolated by treatment with
KOH in ethanol, and Alloc-Cl 2 (Scheme 3). This modified
procedure should enhance the reaction rate over the use of
the oxime plus addition of a base. Indeed, the reaction was
complete in 3 h, affording Alloc-cyanopyridyloxime 9 as a
pale-brownish solid after recrystallization (CH Cl /hexane)
Table 2. Yield of Fmoc-Gly-OH, m.p., formation of byproduct
Fmoc-Gly-Gly-OH.
Yield [%]
Fmoc-Gly-OH
M.p.
[°C]
Purity [%]
Fmoc-Gly-OH
Fmoc-Gly-
Gly-OH [%]
[
a]
[b]
[b]
2
2
3
4
5
6
7
82.9
92.1
48.5
91.6
92.8
166–167
164–165
158–159
165–166
166–167
99.7
99.4
99.7
99.9
99.5
0.17
0.57
0.22
0.01
0.41
in 81% yield and high purity (Ͼ99%), as determined by
HPLC and H NMR spectroscopy.
1
[
a] Authentic m.p. of Fmoc-Gly-OH = 166–167 °C. [b] HPLC
analysis of the crude product.
Table 2 showed, after HPLC analysis of the crude Fmoc-
Gly-OH obtained, that dipeptide formation with analogues
3–7 took place in the range 0.01–0.57% (Figure 1; the pres-
ence of the Fmoc-Gly-Gly-OH dipeptide in the crude mix- Scheme 3. Synthesis of Alloc-cyanopyridyloxime reagent.
ture was identified after co-injection with a pure sample
synthesized in solid-phase) with purities higher than 99%
The performance of novel Alloc-oxime reagent 9 in pre-
in all cases, confirming the first hypothesis about the reac-
venting Alloc-based dipeptide formation was evaluated in
tivity of oxime derivatives. The most reactive oxime-based
the N-protection of H-Gly-OH. In comparison with Fmoc-
introducing reagents, the Alloc-based analogues usually
give rise to higher dimerization percentages as a result of
the considerably lower steric hindrance of the allyl group
than the fluorenylmethyl one, which favors this unwanted
side reaction. The α-amino protection was complete after
reagent 4 gave the highest percentage of the dipeptide
(0.57%) and the least reactive one (i.e., cyanopyridyl, 6)
gave the best results (0.01%). Diester 3 and dicyano 5 also
gave a very similar performance (0.17 and 0.22%, respec-
tively). Yields obtained were reasonably high in general,
with the exception of dicyano analogue 5. The results ob-
tained show that the Fmoc derivative of HOPO (7) is not
recommended due to the formation of a considerable
amount of Fmoc-dipeptide.
1
h in 1%Na CO /dioxane at controlled pH 8, which was
2 3
found to have great impact on the yield obtained. By using
this methodology, Alloc-Gly-OH was afforded in excellent
yield (83%) and purity (99.7%), as determined by HPLC
1
and H NMR spectroscopic analysis. The percentage of di-
peptide in the crude mixture, although higher than in the
analogue Fmoc-protection with compound 6, was ex-
tremely low (0.02%), almost undetectable. This peptidic by-
product was identified after co-injection with a pure dipep-
tide reference, synthesized in the solid-phase by using the
same Alloc-Gly-OH crude (Figure 2).
Figure 1. Fmoc-Gly-OH from 6 and co-injection with an authentic
sample of Fmoc-Gly-Gly-OH.
To extend this methodology to other N-urethane-type
protecting groups, the introduction of the Alloc moiety to
the H-Gly-OH α-amino group was studied (allyloxime car-
bonates have been used in the past for allylation of active
methylene compounds using Pd catalysis).[ Considering
the outstanding performance of cyanopyridyl reagent 6 in
the Fmoc protection compared to other derivatives, only
38]
Figure 2. Alloc-Gly-OH obtained with Alloc-oxime 9 and co-injec-
this analogue was selected to carry out the experiments re- tion with an authentic sample of Alloc-Gly-Gly-OH.
Eur. J. Org. Chem. 2010, 3275–3280
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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3277

S. N. Khattab, R. Subirós-Funosas, A. El-Faham, F. Albericio
FULL PAPER
=
7.7 Hz, 2 H, CH
2
), 7.31 (t, J = 6.9 Hz, 2 H, Ar-H), 7.38–7.43
Conclusions
(
m, 2 H, Ar-H), 7.60 (t, J = 7.7 Hz, 2 H, Ar-H), 7.77 (d, J = 7.7 Hz,
1
3
The use of the novel Fmoc-cyanopyridyloxime 6 and Al- 2 H, Ar-H) ppm. C NMR: δ = 14.04, 14.08, 46.51, 62.57, 62.74,
loc-cyanopyridyloxime 9 allows the preparation of Fmoc- 71.68, 106.71, 120.13, 120.27, 124.87, 127.69, 128.22, 141.41,
amino acids and Alloc-amino acids free of side-products. 142.82, 144.37, 149.50, 151.95, 158.86, 160.22, 160.82 ppm.
C
22
H21NO
7
(411.40): calcd. C 64.23, H 5.14, N 3.40; found C
Thus, Fmoc-Gly-Gly-OH and Alloc-Gly-Gly-OH dipep-
tides are formed in a negligible amount, and the absence of
64.44, H 5.30, N 3.63.
a succinimide moiety precludes the formation of β-alanine Ethyl
2-{[(9H-Fluoren-9-yl)methoxy]carbonyloxyimino}-2-cyano-
acetate (4): The product was obtained as a white solid (m.p. 174–
derivatives. Furthermore, the higher amount of dipeptide
obtained with the cyanoethylester oxime (Oxyma) confirms
1
1
6
75 °C) in 91% yield (3.31 g). H NMR (CDCl
.9 Hz, 3 H, CH ), 4.35 (t, J = 6.9 Hz, 1 H, CH), 4.49 (q, J =
), 4.64 (d, J = 6.9 Hz, 2 H, CH ), 7.35, 7.44 (2 t,
J = 7.7 Hz, 4 H, Ar-H), 7.63, 7.80 (2 d, J = 7.7 Hz, 4 H, Ar-H)
3
): δ = 1.43 (t, J =
3
our previous findings that this leaving group is the best sub-
stitution for hydroxybenzotriazole derivatives.[
32,33]
6.9 Hz, 2 H, CH
2
2
Interest-
ingly and without studying the formation of dipeptides,
Itoh and co-workers proposed a similar oxime, the phenyl
one, for the introduction of the Boc group.[
1
3
ppm. C NMR: δ = 14.09, 46.44, 64.79, 72.62, 106.56, 120.38,
25.23, 127.51, 128.37, 131.23, 141.43, 142.50, 150.86, 156.65 ppm.
(364.35): calcd. C 65.93, H 4.43, N 7.69; found C
6.14, H 4.70, N 7.91.
1
31]
20 16 2 5
C H N O
6
{
[(9H-Fluoren-9-yl)methoxy]carbonyloxy}carbonimidoyl Dicyanide
Experimental Section
(
5): The product was obtained as a white solid (m.p. 150–151 °C)
1
in 87% yield (2.76 g). H NMR (CDCl
CH), 4.54, 4.62 (2 d, J = 7.7 Hz, 2 H, CH
Ar-H), 7.41–7.45 (m, 2 H, Ar-H), 7.59 (t, J = 7.7 Hz, 2 H, Ar-H),
7
1
1
3
): δ = 4.28–4.35 (m, 1 H,
Materials: The solvents used were of HPLC reagent grade. Melting
), 7.32–7.36 (m, 2 H,
points were determined with a Mel-Temp apparatus. Magnetic res-
2
1
13
onance spectra ( H NMR and C NMR spectra) were recorded
with a Joel 500 MHz (Fmoc-oxime experiments) and with a Mer-
cury 400 MHz (Alloc-oxime experiments) spectrometer with chemi-
cal shift values reported in δ units (ppm) relative to an internal
standard. Elemental analyses were performed with a Perkin–Elmer
1
3
.78 (t, J = 6.9 Hz, 2 H, Ar-H) ppm. C NMR: δ = 46.41, 72.60,
06.25, 120.35, 120.39, 125.15, 127.47, 127.52, 128.34, 128.40,
32.63, 141.43, 142.44, 142.49, 150.85, 151.09, 157.36 ppm.
C
6
18
H
11
N
3
O
3
(317.30): calcd. C 68.14, H 3.49, N 13.24; found C
8.39, H 3.61, N 13.48.
2400 elemental analyzer, and the values found were within Ϯ0.3%
of the theoretical values. Follow-up of the reactions and checks of
the purity of the compounds was done by TLC on silica-gel-pro-
tected aluminum sheets (Type 60 GF254, Merck), and the spots
were detected by exposure to UV light at λ = 254 nm for a few
seconds. The compounds were named by using ChemDraw Ultra
version 11, Cambridge Soft Corporation. Exact masses were deter-
mined with a Waters Synapt HDMS mass spectrometer (ESI posi-
tive polarity, W analyzer, 3000 V capillary voltage, 150 and 100 °C
desolvation and source temperature, 40 V sample cone, 100–1500
m/z) by introducing the sample by direct infusion. HPLC analysis
was undertaken by using a reverse-phase Waters 2695 HPLC sepa-
ration module, coupled to a Waters 2998 PDA UV detector, pro-
cessing the chromatograms with Empower software. Separation
N-{[(9H-Fluoren-9-yl)methoxy]carbonyloxy}picolinimidoyl Cyanide
(6): The product was obtained as a white solid (m.p. 165–166 °C)
1
in 93% yield (3.43 g). H NMR (CDCl ): δ = 4.38 (t, J = 7.7 Hz,
3
1 H, CH), 4.62 (d, J = 7.7 Hz, 2 H, CH ), 7.36, 7.44 (2 t, J =
2
6.9 Hz, 4 H, Ar-H), 7.48–7.51 (m, 1 H, Ar-H), 7.66, 7.79 (2 d, J =
7.7 Hz, 4 H, Ar-H), 7.84 (t, J = 6.9 Hz, 1 H, Ar-H), 8.14 (d, J =
1
3
8.4 Hz, 1 H, Ar-H), 8.79 (d, J = 4.6 Hz, 1 H, Ar-H) ppm.
C
NMR: δ = 46.53, 72.00, 107.94, 120.34, 122.08, 125.32, 126.89,
127.48, 128.29, 137.35, 139.67, 141.43, 142.78, 147.16, 150.46,
151.97 ppm. C H N O (369.37): calcd. C 71.54, H 4.09, N 11.38;
2
2
15
3
3
found C 71.77, H 4.32, N 11.53.
9H-Fluoren-9-yl)methyl 2-oxopyridin-1(2H)-yl Carbonate (7): The
product was obtained as a white solid (m.p. 195–196 °C) in 89%
(
was accomplished by using
a
Waters SunFire
C18 (3.5 µ,
1
yield (2.96 g). H NMR (CDCl
4
6
3
): δ = 4.37 (t, J = 7.7 Hz, 1 H, CH),
), 6.20 (t, J = 6.9 Hz, 1 H, Py-H),
.75 (t, J = 9.2 Hz, 1 H, Py-H), 7.33–7.44 (m, 6 H, Ar-H), 7.64 (d,
4.6ϫ100 mm) column and linear gradients of solvent A [0.045%
.61 (d, J = 7.7 Hz, 2 H, CH
2
2
trifluoroacetic acid (TFA) in H O] in solvent B (0.036% TFA in
–1
3
CH CN) with flow = 1.0 mLmin . The mass of peptide materials
1
3
J = 7.7 Hz, 2 H, Ar-H), 7.78 (d, J = 7.7 Hz, 2 H, Ar-H) ppm.
C
was detected by using a HPLC-PDA system as the above described,
coupled to a Waters Micromass ZQ mass detector, with MassLynx
NMR: δ = 46.51, 72.76, 105.27, 120.28, 123.20, 125.33, 127.45,
28.26, 134.89, 139.64, 141.43, 142.67, 152.40, 157.18 ppm.
(333.34): calcd. C 72.06, H 4.54, N 4.20; found C
1
4
.1 software.
General Method for the Preparation of Fmoc-Oxime Derivatives 3–
: A solution of 9-fluorenylmethyloxycarbonyl chloride (10 mmol)
in CH Cl (30 mL) was added slowly to a solution of oxime or 1-
hydroxypyridin-2(1H)-one (10 mmol) and sodium carbonate
20 mmol) in H O (20 mL) with stirring at 0 °C. The resulting clear
mixture was stirred at 0 °C for 30 min and then at room tempera-
ture for 2 h. After dilution with CH Cl (50 mL), the organic phase
was collected and washed with water and saturated aqueous NaCl
30 mL) and then dried with Na SO (anhydrous). After filtering,
the solvent was removed under reduced pressure, and the residue
was recrystallized (CH Cl /hexane) to give Fmoc derivatives 3–7.
Diethyl 2-{[(9H-Fluoren-9-yl)methoxy]carbonyloxyimino}malonate
3): The product was obtained as an oil at room temperature (m.p.
20 4
C H15NO
72.31, H 4.68, N 4.41.
7
N-(9-Fluorenylmethyloxycarbonyl)glycine (Fmoc-Gly-OH): A solu-
tion of Fmoc-OX derivative 3–7 (20 mmol) in acetone (100 mL)
was added dropwise to a stirred solution of glycine (20 mmol) and
2
2
(
2
NaHCO
the reaction mixture was concentrated under reduced pressure and
then extracted with CH Cl (50 mL) to remove the unreacted
3
(50 mmol) in water (100 mL). After stirring overnight,
2
2
2
2
Fmoc-OX derivatives. After cooling, the reaction mixture was
acidified with 10% HCl to congo red litmus paper to give a white
solid, which was filtered and washed with water several times,
dried, and recrystallized (ethyl acetate/n-hexane) to give a white
solid [m.p. 166–167 °C, authentic commercial sample m.p. 166–
167 °C (Table 2)]. The purity of Fmoc-Gly-OH was determined by
injection of 10 µL of a sample prepared from Fmoc-Gly-OH in
acetonitrile onto HPLC by using the following conditions: linear
(
2
4
2
2
(
1
about 20 °C) in 84% yield (3.45 g). H NMR (CDCl
3
): δ = 1.30–
), 4.55 (d, J
1.46 (m, 6 H, 2 CH
3
), 4.29–4.50 (m, 5 H, CH, 2 CH
2
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Novel Reagents for the Introduction of Fmoc and Alloc Protecting Groups
gradient 10 to 90% of solvent A in solvent B over 8 min; PDA
detection at 254 nm. t (Fmoc-Gly-OH) = 7.03 min and co-injec-
tion with an authentic sample of Fmoc-Gly-OH.
washed with CH
layer was acidified to pH 1–2, and the product was extracted with
AcOEt (4ϫ60 mL). The organic layer was dried with Na SO and
2 2
Cl (3ϫ60 mL) at pH 7 and pH 6.5. The aqueous
R
2
4
filtered, and the solvent was removed under reduced pressure to
render a yellow oil in 83% yield (284.1 mg). The purity of Alloc-
Gly-OH was determined by injection onto reverse-phase HPLC by
using the following conditions: linear gradient 5 to 40% of solvent
N-(9-Fluorenylmethyloxycarbonyl)glycinylglycine (Fmoc-Gly-Gly-
OH): The synthesis was carried out in a plastic syringe, attached
to a vacuum manifold so as to effect rapid removal of reagents and
solvent. The 2-chlorotritylchloride resin with a loading 1.55 mmol/
A in solvent B over 8 min; PDA detection at 220 nm. t
R
(Alloc-
g (1 g) was washed with CH
with Fmoc-Gly-OH (2 mmol) and N,N-diisopropylethylamine
DIEA, 14 mmol) in CH Cl (10 mL). Then, the reaction mixture
2 2
Cl (3ϫ50 mL), and then acylated
Gly-OH) = 4.61 min, t (Alloc-Gly-Gly-OH) = 3.86 min. The di-
R
peptide was identified after co-injection with a pure sample, solid-
phase synthesized by using the Alloc-Gly-OH prepared with this
(
2
2
was stirred slowly for 1 min and let to couple for 15 min. Extra
DIEA (6 mmol) was added, and the resin was stirred and left to
stand with stirring from time to time for 45 min. MeOH (1 mL)
was added to the resin, which was stirred for 5 min and then left
to stand for an extra 5 min. The resin was filtered and then washed
1
1
method. H NMR spectroscopy showed a purity above 99%. H
NMR (CD COCD ): δ = 3.88 (d, J = 6.2 Hz, 2 H, CH ), 4.53 (dt,
), 5.14–5.17 (m, 1 H, CH ), 5.28–5.32 (m, 1 H, CH ),
.89–5.94 (m, 1 H, CH), 6.53 (br. s, 1 H, NH) ppm.
3
3
2
2
5
H, CH
2
2
2
N-(Allyloxycarbonyl)glycinylglycine (Alloc-Gly-Gly-OH): The syn-
thesis was carried out in a plastic syringe, attached to a vacuum
manifold so as to effect rapid removal of reagents and solvent. The
with CH
and then deblocked by 20% piperidine in DMF for 7 min, washed
with DMF, CH Cl , and DMF (2ϫ50 mL each), and then coupled
with the next Fmoc-Gly-OH (3 mmol), N,N-diisopropylcarbodiim-
ide (DIC, 3 mmol), and HOBt (3 mmol) for 1 h. The dipeptide was
2 2
Cl and N,N-dimethylformamide (DMF, 2ϫ50 mL each),
2
2
2
-chlorotritylchloride resin (500 mg, loading = 1.55 mmol/g) was
swelled in CH Cl (3ϫ1 mL), conditioned in DMF (5ϫ1 mL),
and then acylated with Fmoc-Gly-OH (0.3 mmol, 1 equiv.) and
DIEA (3 equiv.) in CH Cl (0.8 mL). The reaction mixture was
2
2
cleaved from the resin by TFA/CH
perature for 5 min and then washed with extra solution of TFA/
CH Cl (2%, 20 mL). TFA and CH Cl were removed in vacuo,
2 2
Cl (2%, 50 mL) at room tem-
2
2
manually stirred for 10 min and then extra DIEA (7 equiv.) was
added. The resin was left to stand for 45 min more with stirring.
MeOH (0.4 mL) was added to the resin and this was manually
2
2
2
2
and the crude dipeptide was precipitated with ether. The precipitate
was collected and washed with ether (2ϫ) and then dried under
vacuum. The purity (99.7%) was determined by HPLC analysis by
using a linear gradient 10 to 90% of solvent A in solvent B over
stirred for 15 min. The resin was filtered and washed with CH
2 2
Cl
(
10ϫ1 mL) and DMF (10ϫ1 mL). After deblocking by treatment
with 20% piperidine in DMF (1 + 5 + 5 min) and quantification
of Fmoc, the new loading was calculated (0.57 mmol/g). This H-
8
6
R
min; PDA detection at 254 nm. t (Fmoc-Gly-Gly-OH) =
.29 min.
Gly-resin (101.6 mg) was washed with DMF, CH
10ϫ1 mL each), and it was acylated by using a 0.4  solution of
the Alloc-Gly-OH prepared following the above-mentioned method
3 equiv.) and Oxyma (3 equiv.), preactivated with DIC (3 equiv.)
for 3 min and left to stand for 1 h with stirring. The dipeptide was
cleaved from the resin with 5% TFA/CH Cl (1 mL, 5ϫ5 min) at
room temperature, washing the resin with CH Cl . The solvent and
TFA were removed under an atmosphere of nitrogen, and the crude
dipeptide was precipitated by adding cold Et O (3ϫ3 mL). The
resulting crude was lyophilized in H O (3 mL), obtaining a white
2 2
Cl , and DMF
N-(Allyloxycarbonyloxy)picolinimidoyl Cyanide (9): A solution of
allyl chloroformate (2, 12 mmol) in CH Cl (35 mL) was slowly
added to a solution of the potassium salt of N-hydroxypicolin-
(
2
2
(
imidoyl cyanide (8, 12 mmol)[ in H
°C. The resulting biphasic mixture was vigorously stirred at 0 °C
for 30 min and then at room temperature for 3 h. After dilution
with CH Cl (50 mL), the organic phase was collected, washed with
water (2ϫ50 mL) and saturated aqueous NaCl (2ϫ50 mL), and
dried with anhydrous Mg SO . After filtering, the solvent was re-
moved under reduced pressure, and the residue was recrystallized
CH Cl /hexane) to give desired Alloc-oxime compound 9 as a pale
brownish solid (m.p. 102–103 °C) in 81% yield (2.24 g). H NMR
CDCl ): δ = 4.85–4.86 (dt, 2 H, CH ), 5.38–5.40 (m, 1 H, CH ),
.46–5.50 (m, 1 H, CH ), 5.97–6.07 (m, 1 H, CH), 7.47–7.50 (m, 1
H, Py-H), 7.82–7.86 (m, 1 H, Py-H), 8.12–8.15 (m, 1 H, Py-H),
33]
O (40 mL) with stirring at
2
0
2
2
2
2
2
2
2
2
4
2
solid powder in 92% yield (11.5 mg). The purity (Ͼ99%) was deter-
mined by HPLC analysis by using a linear gradient 5 to 100% of
(
2
2
1
solvent A in solvent B over 8 min; PDA detection at 220 nm. t
Alloc-Gly-Gly-OH) = 3.39 min. HPLC-MS showed the expected
R
(
5
3
2
2
(
+
2
mass for the dipeptide: m/z = [M + H] = 217.04.
8
1
1
.77–8.79 (m, 1 H, Py-H) ppm. 13C NMR (CCl
3
): δ = 70.62, 107.98,
20.85, 122.16, 126.88, 130.48, 137.39, 139.63, 147.34, 150.52, Acknowledgments
51.89 ppm. The purity of 9 was also determined after injection
onto reverse-phase HPLC by using the following conditions: linear
This work was partially supported by the Comisión Interde-
gradient 5 to 100% of solvent A in solvent B over 8 min; PDA partamental de Científica y Tecnológica (CICYT) (CTQ2009-
detection at 220 nm. t
anide] = 6.84 min. A sample for exact mass determination was pre-
pared, dissolving in CHCl and diluting 1 to 100 in H O/MeOH
1:1): m/z = 254.0546 [M + Na] .
R
[N-(allyloxycarbonyloxy)picolinimidoyl cy-
07758), the Generalitat de Catalunya (2009SGR 1024), the Institute
for Research in Biomedicine, and the Barcelona Science Park.
Suzhou Highfine Biotech Co., Ltd. is thanked for a sample of the
Fmoc-oxime.
3
2
+
(
N-(Allyloxycarbonyl)glycine (Alloc-Gly-OH):
2.16 mmol) in dioxane (7 mL) was added dropwise to a stirred
solution of H-Gly-OH (2.37 mmol) in 1% aqueous Na CO
7 mL). The pH of the reaction mixture was controlled at 8 with
addition of 10% aqueous Na CO . After stirring the reaction mix-
ture for 1 h at room temperature, H O (50 mL) was added. The
resulting mixture was acidified to pH 7 by addition of 2% aqueous
HCl, and the white solid N-hydroxypicolinimidoyl cyanide was fil-
tered. To remove the remaining oxime, the aqueous layer was
A solution of 9
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3279

S. N. Khattab, R. Subirós-Funosas, A. El-Faham, F. Albericio
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Received: January 10, 2010
Published Online: May 4, 2010
3280
www.eurjoc.org
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 3275–3280