Nα-Fmoc-protected ω-azido- and ω-alkynyl-L- amino acids as building blocks for the synthesis of "clickable" peptides

Article abstract of DOI:10.1002/ejoc.200800717

The growing interest in the 1,4-disubstituted-1,2,3-triazolyl moiety as an amide bond surrogate and its formation through very mild, chemoselective, and bioorthogonal Cu^I-catalyzed Huisgen 1,3-dipolar [3+2] cycloaddition of an alkynyl to an azi

Full text of DOI:10.1002/ejoc.200800717

FULL PAPER
DOI: 10.1002/ejoc.200800717
Nα-Fmoc-Protected ω-Azido- and ω-Alkynyl-
-amino Acids as Building Blocks
L
for the Synthesis of “Clickable” Peptides
Alexandra Le Chevalier Isaad,[a,b] Francesca Barbetti,[c] Paolo Rovero,[a,d]
Anna Maria D’Ursi,[e] Mario Chelli,[a,b] Michael Chorev,[f,g] and Anna Maria Papini*[a,b]
Keywords: Amino acids / Modified amino acids / Azides / Alkynes / Solid-phase peptide synthesis / Click chemistry
The growing interest in the 1,4-disubstituted-1,2,3-triazolyl
moiety as an amide bond surrogate and its formation through
very mild, chemoselective, and bioorthogonal CuI-catalyzed
Huisgen 1,3-dipolar [3+2] cycloaddition of an alkynyl to an
azido function, presented an unmet need for specifically de-
droxy precursors by the respective diazo-transfer reactions
and sequential nucleophilic substitutions initially by halide
followed by azide. The homologous Nα-Fmoc-ω-yne-
L-amino
acids were prepared by alkylation of a chiral auxiliary, which
is a NiII complex of Schiff base derived from glycine and (S)-
signed α-amino-acid-derived building blocks. Herein we re- 2-(N-benzylprolyl)aminobenzophenone, with ω-bromoal-
port the synthesis of unnatural homologous series of Nα-
Fmoc-protected ω-yne- and ω-azido- -amino acids compati-
ble with the Fmoc/tBu-based solid-phase peptide synthesis.
These building blocks can be incorporated into pseudopep-
tides that can serve as precursors of inter- and intramolecular
click reactions. The homologous Nα-Fmoc-ω-azido-
L-amino
kynes. These building blocks will be instrumental for con-
structing side-chain modified peptides, interside-chain pep-
tide chimera, peptide small molecule conjugates, and cyclo-
peptides, which were cyclized through 1,4-disubstituted
1,2,3-triazolyl-containing side-chain-to-side-chain bridges.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2008)
L
acids were prepared from either the ω-amino or the ω-hy-
of post-translational modifications. To this end there are
several reports in which non-coded α-amino acids modified
by ω-azido and ω-alkyne functionalities were used for bio-
orthogonal introduction of reporters in peptides and pro-
teins.[5–7]
Introduction
Endogenous as well as exogenous post-translational
modifications of proteins and peptides are recognized as
important means to alter bioactivity profiles, recognition,
metabolism, immunogenicity, physicochemical properties,
and to introduce affinity tags and labels. Methods such as
expression of proteins containing non-coded amino acids,[1]
native and expressed chemical ligations,[2] employment of
bioorthogonal chemical reporters and reactions,[3] and in-
troduction of genetically encoded functional tags[4] present
the different approaches explored for specific introduction
The unique chemoselectivity of the alkyne and azido
functionalities results in a very high degree of bioorthogon-
ality.[5–9] This is characterized by their high stability in
water and lack of reactivity toward all naturally abundant
functional groups that may be present in natural biopoly-
mers.[5–9] The prototypical click reaction, the so called CuI-
catalyzed Huisgen 1,3-dipolar [3+2] cycloaddition between
an azido and a terminal alkynyl function generates the 1,4-
disubstituted-1,2,3-triazolyl moiety. It is carried out at
room temperature, tolerates the presence of water and re-
sults in high yields and excellent regioselectivity.[10–12]
The ω-azido- and ω-alkynyl-α-amino acids are used to
enable modifications such as intramolecular side-chain-to-
side-chain cyclizations,[8–13] macrocyclizations,[6e] conjuga-
tion of carbohydrates to amino acids[5] and peptides,[6j] gen-
eration of other bioconjugates,[14] and incorporation into
recombinant proteins by either the mutated methionyl-
tRNA synthetase[15] or the orthogonal amber tRNA/tRNA
synthetase pair.[6d,16] To date, the diazo-transfer reaction is
frequently used to generate α-azido-acids in solution[17,18]
and α-azido-peptides on solid support.[17] However, ω-
azido-α-amino acids such as β-azidoalanine were prepared
[a] Laboratory of Peptide & Protein Chemistry & Biology, Polo
Scientifico e Tecnologico, University of Firenze,
50019 Sesto Fiorentino, Italy
Fax: +39-055-457-3584
E-mail: annamaria.papini@unifi.it
[b] Dipartimento di Chimica Organica and CNR-ICCOM, Polo
Scientifico e Tecnologico, University of Firenze
Via della Lastruccia 13, 50019 Sesto Fiorentino, Italy
[c] Toscana Biomarkers Srl
Via Fiorentina 1, 53100 Siena, Italy
[d] Dipartimento di Scienze Farmaceutiche, Polo Scientifico e Tec-
nologico, University of Firenze,
Via Ugo Schiff 3, 50019 Sesto Fiorentino, Italy
[e] Dipartimento di Scienze Farmaceutiche, University of Salerno,
Via Ponte Don Melillo 11c, 84089 Salerno, Italy
[f] Laboratory for Translational Research, Harvard Medical
School,
One Kendal Square, Building 600, Cambridge, MA 02139, USA
[g] Department of Medicine, Brigham and Women’s Hospital,
75 Francis Street, Boston, MA 02115, USA
5308
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Building Blocks for the Synthesis of “Clickable” Peptides
from either the salt of α-amino-β-propiolactone,[19] the pro-
tected serinol[5] or diazo-transfer reaction on the Nα-Fmoc-
protected α,β-diaminopropionic acid.[9] The ω-alkynyl-α-
amino acids, in contrast to the commercially available pro-
pargylglycine, such as the racemic 2-aminohexynoic acid
and the optically active 2-aminopentynoic acid were pre-
pared from diethyl (acetylamino)malonate[20] and protected
homoserinal,[5] respectively. Evidently, there is an unmet
need for syntheses that will furnish a series of homologous
enantiomerically pure Nα-protected ω-azido- and ω-alk-
ynyl-α-amino acids as building blocks for the rapidly ex-
panding array of applications mentioned above.
Results and Discussion
Our preferred synthetic strategy was to take advantage
whenever possible of the pre-defined chiralities of commer-
cially available α-amino acid derivatives and use them as
the starting materials in the synthesis of the ω-azido- and
ω-alkynyl-α-amino acids. An efficient and convenient meth-
odology for generating organic azides is CuII-catalyzed di-
azo transfer from amines by trifluoromethanesulfonyl az-
ide.[9,21] In this manner Nα-Boc--ornithine and Nα-Boc--
lysine were converted in good yield into the respective Nα-
Fmoc-δ-azido--norvaline (9) and Nα-Fmoc-ε-azido--nor-
leucine (10) without affecting the original chirality.
Recently, we reported the use of Nα-Fmoc--Nle(ε-N3)-
OH together with Nα-Fmoc--propargylglycine (Nα-Fmoc-
-Pra-OH) in the synthesis of cyclic peptide via i-to-(i+4)
intramolecular side-chain-to-side-chain azido-to-alkynyl
CuI-catalyzed Huisgen 1,3-dipolar [3+2] cycloaddition gen-
erating a topologic mimic of the helical structure stabilized
by the corresponding i-to-(i+4) intramolecular side-chain-
to-side-chain lactam-bridged cyclopeptide.[13]
Herein we report the development of efficient and conve-
nient synthetic pathways to non-coded Nα-Fmoc-ω-azido-
and Nα-Fmoc-ω-alkynyl--amino acids suitable for Fmoc/
tBu solid-phase peptide synthesis (SPPS) (Figure 1).
The conversion of -hSer into Nα-Fmoc-amino-γ-azido-
-butyric acid (8) required a multi-step strategy. The pre-
viously reported synthesis of Nα-Boc--Abu(γ-N3)-OMe (5)
started from the -hSer-derived N-protected δ-lactone. It in-
cluded one-pot ring-opening/substitution reaction to gener-
ating the hydrobromide of α-amino-γ-bromo--butyric acid
that was then transformed into the corresponding γ-iodo
derivative and subsequently to the desired azide.[22] Our
synthesis of 5 starts with the direct conversion of the unpro-
tected -hSer to α-amino-γ-bromo--butyric acid·HBr (2a)
(Scheme 1). After saponification of Nα-Boc--Abu(γ-N3)-
OMe (5), we removed the Boc group and replaced it with
Fmoc to obtain the desired Nα-Fmoc--Abu(γ-N3)-OH (8).
This multi-step synthesis was carried out without purifica-
tion of intermediates and the progress of the reactions was
1
monitored by H NMR following the characteristic chemi-
cal shifts of the γ-H2 and the presence of the protecting
groups.
The strategy to synthesize ω-alkynyl--amino acid homo-
logues higher than propargylglycine containing (CH2)n
(where n = 3 and 4) in the side chain employed asymmetric
Figure 1. Nα-Fmoc-ω-azido- and Nα-Fmoc-ω-alkynyl--amino ac-
ids as building blocks for Fmoc/tBu solid-phase peptide synthesis. synthesis to elaborate glycine used as the starting material.
Scheme 1. Synthesis of Nα-Fmoc--Abu(γ-N3)-OH.
Eur. J. Org. Chem. 2008, 5308–5314
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A. M. Papini et al.
FULL PAPER
Scheme 2. Asymmetric synthesis of (S)-ω-alkynyl-α-amino acids employing the chiral auxiliary [NiII-(S)BPB-Gly] (12).
The NiII complex [NiII-(S)BPB-Gly][23] (12) of the Schiff tiomerically pure Nα-Fmoc-protected ω-alkynyl- and ω-
base derived from glycine and (S)-2-(N-benzylprolyl)amino- azido--amino acids in which the alkynyl and azido func-
benzophenone (BPB) (11), was used as a chiral auxiliary tions are carried on side-chains of variable length (2–4
during the Cα-alkylations.[24,25] Alkylations through the si- methylene groups). These synthetic strategies are suitable
face of the glycine enolate are largely favored leading to for scaling up and are generally applicable for the prepara-
high enantiomeric excess of the (S)-ω-alkynyl-α-amino acid tion of higher homologs. In addition, following the reported
residue containing diastereoisomer (Scheme 2).[25]
syntheses we generate not only the Nα-Fmoc- but also Nα-
Cα-Alkylation of the glycine moiety, incorporated into Boc-protected -amino acids thus providing building blocks
the chiral auxiliary [NiII-(S)BPB-Gly] (12), by excess of ω- for orthogonal methodologies in solid-phase peptide syn-
bromoalkynes (1.4 equiv.) 13 and 14 was carried out in an- thesis. Extensive application of these enantiomerically pure
hydrous acetonitrile in the presence of NaOH yielding the building blocks in syntheses of side-chain-modified pep-
corresponding Cα-alkylated complexes 15 and 16 in good tides, inter-side chain peptide–peptide, and peptide–carbo-
diastereomeric excess (monitored by Ultra Performance Li- hydrate chimera, peptide–small molecule conjugates and bio-
quid Chromatography, Table 1). The ω-bromoalkynes 13 conjugates, and linear precursors of side-chain-to-side-
and 14 were prepared from the corresponding alcohols by chain-bridged cyclopeptides is in progress in our laborato-
treatment with TsCl followed by LiBr.
ries.
Table 1. Diastereomeric ratio of the ω-alkylated complexes.
Products of alkylation of the
Diastereomeric ratio
(SS)/(SR)
Experimental Section
[NiII-(S)BPB-Gly] complex with
The chemicals were purchased from Sigma–Aldrich and used with-
out further purification. Protected amino acids were obtained from
Novabiochem AG (Laufelfingen, Switzerland). TLC were carried
out on silica gel precoated plates (Merck; 60 Å F254) and spots
located with: (a) UV light (254 and 366 nm), (b) ninhydrin (solu-
tion in acetone), (c) Cl2/tolidine, (d) fluorescamine, (e) I2, (f) a basic
solution of permanganate [KMnO4 (3 g), K2CO3 (20 g), and
NaOH (0.25 g) in water (300 mL)]. Flash Column Chromatography
(FCC) was performed on Merck silica gel 60 (230–400 mesh) ac-
cording to Still et al.[26]
5-Bromopent-1-yne (13)
6-Bromohex-1-yne (14)
71:29
88:12
The straightforward separation of the diastereoisomeric
mixture by FCC was followed by short hydrolysis of the
pure diastereomeric alkylated complexes 15 and 16 in 2 
HCl. Treatment of the crude hydrolysis products with H+
Chelex resin removed the residual NiII, which was found to
interfere in the subsequent Nα-protection by Fmoc-OSu.
Finally, the free ω-alkynyl-α--amino acids 17 and 18
were Nα-protected as Fmoc to yield the building blocks 19
and 20 in adequate quantities for Fmoc/tBu solid-phase
peptide synthesis.
1H and 13C NMR spectra were recorded at 400 and 100 MHz,
respectively, on a Varian spectrometer in deuterated chloroform
solution and are reported in parts per million (ppm), with solvent
resonance used as reference. Melting point was determined on a
Büchi mod. 510 apparatus and are uncorrected. Elemental analysis
was performed on a Perkin–Elmer 240 C Elemental Analyzer. In-
frared spectra were recorded on a Perkin–Elmer mod. BX II FT-
IR spectrometer. The [α]D were obtained on Perkin–Elmer mod.
343 Polarimeter in cell of 1 dm. Products were analyzed by ACQU-
Conclusions
Herein we report efficient synthetic pathways for the con-
struction of building blocks comprised of a series of enan- ITY UPLC (Waters Corporation, Milford, Massachusetts) coupled
5310 Eur. J. Org. Chem. 2008, 5308–5314
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© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Building Blocks for the Synthesis of “Clickable” Peptides
to single quadrupole ESCI-MS (Micromass ZQ) using
2.1ϫ50 mm 1.7 µm ACQUITY BEH C18 at 30 °C, with a flow
rate of 0.45 mL/min. The solvent systems used were A (0.1% TFA
in H2O) and B (0.1% TFA in CH3CN).
a
a
ously stirred mixture of NaN3 (1.775 g, 27.3 mmol) in H2O
(4.5 mL) and CH2Cl2 (7.5 mL) at 0 °C. The resulting mixture was
warmed to room temp. and stirring was continued for 2 h. The
water layer was extracted with CH2Cl2 (2ϫ3 mL) and the com-
bined organic layers were washed with saturated aqueous Na2CO3
(8.5 mL). The resulting solution of TfN3 in CH2Cl2 was then slowly
added to a solution of Nα-Boc--norvaline (669 mg, 2.88 mmol),
K2CO3 (600 mg, 4.32 mmol), and CuSO4·5H2O (7 mg, 0.028
mmol) in H2O (8 mL) and MeOH (17 mL). The mixture was stirred
overnight and the reaction was monitored by TLC: Rf(a & b) = 0.76
(iPrOH/AcOEt/H2O, 6:1:3). The organic phase was evaporated un-
der vacuum. The water layer was acidified to pH 6 with concd.
HCl, diluted with 0.25  of phosphate buffer at pH 6.2 (25 mL),
and extracted with CH2Cl2 (4ϫ50 mL). The organic layers were
washed with brine (25 mL), dried with anhydrous Na2SO4, filtered
and the solvent was evaporated under vacuum. The crude colorless
oil was purified on a RP-18 LiChroprep column employing a step-
gradient of CH3CN in H2O to afford the Nα-Boc--Abu(δ-N3)-OH
Methyl 4-Azido-2S-{[(1,1-dimethylethoxy)carbonyl]amino}butyrate
[Nα-Boc- -Abu(γ-N3)-OMe] (5): Nα-Boc--Abu(γ-I)-OMe (4) (pre-
L
pared as reported in Scheme 1) (2.58 g, 7.52 mmol) in anhydrous
DMF (30 mL) was added to NaN3 (0.846 g, 13.01 mmol) under
nitrogen. The mixture was heated at 90 °C for 2 h and then poured
into an ice/water mixture (180 mL). The aqueous layer was ex-
tracted with AcOEt (60 mL). The organic phase was washed with
brine, dried on anhyd Na2SO4, filtered, and evaporated to obtain
crude azide as a yellow oil (0.929 g, 48%). 1H NMR (CDCl3,
200 MHz): δ = 5.40 (br. s, 1 H, NH), 4.26 (br. s, 1 H, α-H), 3.64
(s, 3 H, OCH3), 3.31 (t, J = 6.6 Hz, 2 H, γ-H2), 2.10–1.64 (m, 2 H,
β-H ), 1.33 (s, 9 H, tBu) ppm. IR (KBr): ν = 2101 (N ) cm–1.
˜
2
3
4-Azido-2S-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}butanoic Acid
[Nα-Fmoc-
-Abu(γ-N3)-OH] (8): Cleavage of the Boc protecting
(324 mg, 44%). IR (CHCl ): ν = 2101 (N ) cm–1. ESI-MS: calcd.
˜
3
3
L
for C10H18N4O4 [M + Na]+ 281.12; found 281.02. [α]D = –1.8 (c =
1.0, MeOH). 1H NMR (400 MHz, CDCl3): δ = 9.10 (br. s, 1 H,
COOH), 6.58 (br. s, 1 H, NH), 4.34–4.17 (m, 1 H, α-H), 3.33 (t, J
= 6.8 Hz, 2 H, δ-H2), 1.99–1.65 (m, 4 H, 2ϫ CH2), 1.45 (s, 9 H,
tBu) ppm. 13C NMR (50 MHz, CDCl3): δ = 176.71 (COOH),
155.58 (CONH), 80.56 (Me3C), 52.85 (C-α), 50.82 (C-δ), 29.70
(CH2), 28.27 (3ϫ CH3), 24.90 (CH2) ppm. C10H18N4O4 (258.27):
calcd. C 46.50, H 7.02, N 21.69; found C 46.48, H 7.05, N 21.71.
group of Nα-Boc--Abu(γ-N3)-OMe (5) (0.748 g, 2.9 mmol) was
achieved by treatment with an excess of TFA (10 mL) at room
temp. for 10 min. The reaction was checked by TLC: Rf(a) = 0.10
(AcOEt/n-hexane, 1:1). TFA was removed by flushing with N2 and
the residue dissolved in water and lyophilized. The methyl ester was
hydrolyzed by stirring with 1  NaOH (5 mL) at room temp. for
6 h. The solution was then treated with concd HCl until pH 7 and
lyophilized to afford the free amino acid H--Abu(γ-N3)-OH. A
solution of Fmoc-OSu (0.843 g, 2.5 mmol) in dioxane (6 mL) was
added dropwise to a solution of the deprotected amino acid in di-
oxane (10 mL). A solution of 1  NaOH was subsequently slowly
added until pH 8 and the reaction mixture was stirred at room
temp. for 3 h. The reaction was monitored by TLC: Rf(a) = 0.58
(CH2Cl2/MeOH, 9:2). Water (7.5 mL) was added and the solution
was acidified with concd HCl until pH 3. The product was ex-
tracted with CH2Cl2 (3ϫ20 mL), dried with anhyd Na2SO4, fil-
tered and the solvent evaporated under vacuum. The crude was
purified by FCC on silica gel employing a step-gradient of MeOH
in CH2Cl2, 0%-10% to obtain the Nα-Fmoc--Abu(γ-N3)-OH
(124 mg, 27%) as yellow oil. RP-UPLC: Rt = 1.31 min (50 to 100%
6-Azido-2S-{[(1,1-dimethylethoxy)carbonyl]amino}hexanoic
Acid
[Nα-Boc-
L-Nle(ε-N3)-OH] (7): Tf2O (1.35 mL, 8.13 mmol) was
added dropwise to a vigorously stirred mixture of NaN3 (2.635 g,
40.5 mmol) in H2O (6.5 mL) and CH2Cl2 (11 mL) at 0 °C. The re-
sulting mixture was warmed to room temp. and stirring was contin-
ued for 2 h. The water layer was extracted with CH2Cl2 (2ϫ4 mL)
and the combined organic layers were washed with saturated aque-
ous Na2CO3 (12.5 mL). The resulting solution of TfN3 in CH2Cl2
was then slowly added to a solution of Nα-Boc-lysine (1.0 g,
4.06 mmol), K2CO3 (0.84 g, 6.08 mmol), and CuSO4·5H2O (10 mg,
0.04 mmol) in H2O (13 mL) and MeOH (27 mL). The mixture was
stirred overnight and the reaction was monitored by TLC: Rf(a & b)
= 0.81 (iPrOH/AcOEt/H2O, 6:1:3). The organic phase was evapo-
rated under vacuum. The water layer was acidified to pH 6 with
concd. HCl, diluted with 0.25  phosphate buffer at pH 6.2
(25 mL), and extracted with CH2Cl2 (4ϫ50 mL). The organic lay-
ers were washed with brine (25 mL), dried with anhydrous Na2SO4,
filtered and the solvent was evaporated under vacuum. The color-
less oil was purified on a RP-18 LiChroprep column using a step-
gradient of CH3CN in H2O to afford Nα-Boc--Nle(ε-N3)-OH
of B in 3 min). IR (CHCl ): ν = 2100 (N ) cm–1. ESI-MS: calcd.
˜
3
3
for C19H18N4O4 [M + Na]+ 389.12; found 389.4. [α]D = –11.5 (c =
1
1.0, MeOH). H NMR (400 MHz, CDCl3): δ = 7.75 (pseudo d, J
= 7.6 Hz, 2 H, fluorenyl 4-H and 5-H), 7.54 (pseudo d, J = 7.4 Hz,
2 H, fluorenyl 1-H and 8-H), 7.39 (pseudo t, 2 H, fluorenyl 3-H
and 6-H), 7.31 (pseudo t, 2 H, fluorenyl 2-H and 7-H), 6.14 (br. s,
COOH), 5.63 (m, 1 H, NH), 4.53–4.41 (m, 3 H, CH2-O and α-H),
4.21 (t, J = 6.8 Hz, 1 H, fluorenyl 9-H), 3.42–3.39 (m, 2 H, γ-H2),
2.19–1.96 (m, 2 H, β-H2) ppm. 13C NMR (100 MHz, CDCl3): δ =
172.71 (COOH), 156.26 (CONH), 143.53 and 141.29 (fluorenyl C-
4a, C-4b, C-8a, and C-9a), 127.76, 127.08, 125.04, 124.99 (fluorenyl
C-2 to C-7), 120.00 (fluorenyl C-1 and C-8), 67.17 (CH2-O), 51.70
(C-α), 47.68 (C-γ), 47.09 (fluorenyl C-9), 31.21 (C-β) ppm.
C19H18N4O4 (366.37): calcd. C 62.29, H 4.95, N 15.29; found C
62.36, H 4.99, N 15.24.
(451 mg, 41%). IR (CHCl ): ν = 2100 (N ) cm–1. ESI-MS: calcd.
˜
3
3
for C11H20N4O4 [M – H]– 271.14; found 271.06. [α]D = –1.1 (c =
1.0, MeOH). 1H NMR (400 MHz, CDCl3): δ = 8.93 (br. s, 1 H,
COOH), 6.50 (br. s, 1 H, NH), 4.31–4.14 (m, 1 H, α-H), 3.28 (t, J
= 6.8 Hz, 2 H, ε-H2), 1.90–1.48 (m, 6 H, 3ϫ CH2), 1.45 (s, 9 H,
tBu) ppm. 13C NMR (50 MHz, CDCl3): δ = 177.14 (COOH),
155.53 (CONH), 80.32 (Me3C), 54.29 (C-α), 51.17 (C-ε), 31.95
(CH2), 28.42 (CH2), 28.30 (3ϫ CH3), 22.55 (CH2) ppm.
C11H20N4O4 (272.30): calcd. C 48.52, H 7.40, N 20.58; found C
48.56, H 7.44, N 20.61.
5-Azido-2S-{[(1,1-dimethylethoxy)carbonyl]amino}pentanoic Acid
[Nα-Boc- -Nva(δ-N3)-OH] (6): Nα-Boc--Orn(Nδ-Cbz)-OH (1.0 g,
L
2.73 mmol) in MeOH (25 mL) was treated with H2 and 10% Pd/C
(0.1 g, 0.094 mmol Pd) at atmospheric pressure for 16 h. The mix-
ture was filtered through Celite and washed with MeOH. After
solvent evaporation, the deprotected Nα-Boc--ornithine was ob-
tained as a white powder (669 mg) that was used without further
purification. A solution of TfN3 in CH2Cl2, was obtained as fol-
lows: Tf2O (0.92 mL, 5.54 mmol) was added dropwise to a vigor-
General Procedure for the Synthesis of Nα-Fmoc-
(9) and Nα-Fmoc-
L
-Nva(δ-N3)-OH
L
-Nle(ε-N3)-OH (10): Cleavage of the Boc protect-
ing group of Nα-Boc-ω-azido--α-amino acids 6 and 7 (3.96 mmol)
was achieved by treatment with an excess of concd. HCl (2.5 mL)
at room temp. for 6 h. The residue was dissolved in water (5 mL)
and lyophilized. A solution of (2,5-dioxo-1-pyrrolidinyl) (9H-flu-
Eur. J. Org. Chem. 2008, 5308–5314
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A. M. Papini et al.
FULL PAPER
oren-9-ylmethyl) carbonate (Fmoc-OSu, 4.36 mmol) in dioxane
(20 mL) was then added dropwise to a stirred solution of the depro-
tected amino acid in dioxane (30 mL). A solution of 1  NaOH
was subsequently slowly added until pH 8–9 and the reaction mix-
ture was stirred at room temp. for 2.5 h. The reaction was moni-
tored by TLC (CH2Cl2/MeOH, 9:2). Water (12 mL) was added and
the solution was acidified with 2  HCl until pH 3. The product
was extracted with CH2Cl2 (3ϫ30 mL), dried with anhydrous
Na2SO4, filtered and the solvent removed under vacuum. The crude
material was purified by FCC on silica gel (employing a step-gradi-
ent of MeOH in CH2Cl2, 0–10%) to obtain the corresponding pure
Nα-Fmoc--ω-azido-α-amino acids 9 and 10 as yellow oils.
material was dried under vacuum to afford BPB (1.873 g, 29%).
NMR spectroscopic data were in accordance with the literature.[27]
ESI-MS: calcd. for C25H24N2O2 [M + H]+: 385.19; found 385.2.
1H NMR (400 MHz, CDCl3): δ = 11.52 (s, 1 H, NH), 8.56 (d, J =
8.4 Hz, 1 H, ArH), 7.79–7.36 (m, 9 H, ArH), 7.15 (m, 4 H, ArH),
δA = 3.92, δB = 3.59 (syst AB, JAB = 12.8 Hz, 2 H, PhCH2), 3.32
(dd, Jα,β = 4.4, Jα,βЈ = 10.0 Hz, 1 H, α-H), 3.22 (dd, Jδ,δЈ = Jδ,γ
=
6.4 Hz, 1 H, δ-H), 2.41 (dd, Jβ,βЈ = 8.8, Jβ,γ = 16 Hz, 1 H, βЈ-H),
2.26 (ddd, JδδЈ = 6.4, Jδγ = 12.8, JδγЈ = 22 Hz, 1 H, δЈ-H), 1.96
(ddd, Jβ,βЈ = 8.8, Jβ,γ = 4.4, JβγЈ = 16.4 Hz, 1 H, β-H), 1.85–1.76
(m, 2 H, γ-H and γЈ-H) ppm. 13C NMR (100 MHz, CDCl3): δ =
198.03 (Ph-CO-Ph), 174.64 (N-C=O), 139.16, 138.54, 138.12,
133.37, 132.55, 132.48, 130.11, 129.12, 128.30, 128.15, 127.05,
125.32, 122.19, 121.52 (18 Ar), 68.25 (C-α), 59.82 (PhCH2), 53.85
(C-δ), 30.98 (C-β), 24.14 (C-γ) ppm.
5-Azido-2S-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}pentanoic
Acid [Nα-Fmoc-
L
-Nva(δ-N3)-OH] (9): Yield 69%. TLC: Rf(a) = 0.58
(CH2Cl2/MeOH; 9:2). RP-UPLC: Rt = 1.37 min (50 to 100% of B
in 3 min). IR (CHCl ): ν = 2100 (N ) cm–1. ESI-MS: calcd. for General Procedure for the Alkylation of [NiII-(S)BPB-Gly] Complex
˜
3
3
C20H20N4O4 [M + Na]+ 403.14; found 403.3. [α]D = –2.3 (c = 1.0, with ω-Alkynyl Bromides: To a stirred mixture of [NiII-(S)BPB-Gly]
MeOH). 1H NMR (400 MHz, CDCl3): δ = 7.76 (d, J3,4 = J5,6
7.6 Hz, 2 H, fluorenyl 4-H and 5-H), 7.61 (pseudo d, J1,2 = J7,8
=
=
(12) (prepared from BPB following the procedure previously de-
scribed)[23] (1.99 g, 4 mmol) in anhydrous CH3CN (17.5 mL) were
added, under N2, finely powdered NaOH (0.4 g, 10 mmol) and ω-
alkynyl bromide (6.01 mmol). After 5 h, the reaction mixture was
treated with 0.1  HCl (59 mL) and the red product extracted with
CH2Cl2 (4ϫ40 mL), dried with MgSO4, the mixture was filtered
and the solvent removed under vacuum. The crude was purified by
FCC on silica gel (CH2Cl2/Me2CO, 2:1) affording the product as a
7.6 Hz, 2 H, fluorenyl 1-H and 8-H), 7.40 (pseudo t, 2 H, fluorenyl
3-H and 6-H), 7.31 (pseudo t, 2 H, fluorenyl 2-H and 7-H), 6.16
(br. s, COOH), 5.34 (m, 1 H, NH), 4.45–4.40 (m, 3 H, CH2-O and
α-H), 4.22 (t, J = 6.6 Hz, 1 H, fluorenyl 9-H), 3.37–3.30 (m, 2 H,
δ-H2), 2.01–1.46 (m, 4 H, 2ϫ CH2) ppm. 13C NMR (100 MHz,
CDCl3): δ = 175.72 (COOH), 156.72 (CONH), 143.75, 143.57, and
141.33 (fluorenyl C-4a, C-4b, C-8a, and C-9a), 127.76, 127.08, and red amorphous solid.
125.00 (fluorenyl C-2 to C-7), 120.02 (fluorenyl C-1 and C-8), 67.12
Alkylation of [NiII-(S)BPB-Gly] Complex with 5-Bromopent-1-yne
(CH2-O), 53.16 (C-α), 50.76 (C-δ), 47.15 (fluorenyl C-9), 29.62
(CH2), 24.81 (CH2) ppm. C20H20N4O4 (380.40): calcd. C 63.15, H
5.30, N 14.73; found C 63.09, H 5.25, N 14.80.
(13): Yield of 15: 59%. M.p. 83.5–85 °C; TLC: Rf(a & e) = 0.57
[CH2Cl2/Me2CO, 2:1]. [α]D = –1522 (c = 0.5, MeOH). ESI-MS:
calcd. for C32H31N3NiO3 [M + H]+: 564.18; found 564.5. 1H NMR
(400 MHz, CDCl3): δ = 8.12 (d, J = 8.4 Hz, 1 H, ArH), 8.04 (d, J
= 7.6 Hz, 2 H, ArH), 7.50–7.41 (m, 3 H, ArH), 7.33 (t, J = 7.6 Hz,
2 H, ArH), 7.25–7.10 (m, 3 H, ArH), 6.95 (d, J = 7.6 Hz, 1 H,
6-Azido-2S-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}hexanoic
Acid [Nα-Fmoc-
L-Nle(ε-N3)-OH] (10): Yield 64%. TLC: Rf(a) = 0.58
(CH2Cl2/MeOH, 9:2). RP-UPLC: Rt = 1.51 min (50 to 100% of B
in 3 min). IR (CHCl ): ν = 2100 (N ) cm–1. ESI-MS: calcd. for ArH), 6.64–6.60 (m, 2 H, ArH), 4.43 (d, J = 12.6 Hz, 1 H, PhCH2),
˜
3
3
C21H22N4O4 [M + Na]+ 417.15; found 417.2. [α]D = –2.5 (c = 1.0, 3.86 (dd, J1 = 3.4; J2 = 8.8 Hz, 1 H, Pro α-H), 3.56 (d, J = 12.6 Hz,
MeOH). 1H NMR (400 MHz, CDCl3): δ = 7.74 (d, J3,4 = J5,6
7.4 Hz, 2 H, fluorenyl 4-H and 5-H), 7.54 (d, J1,2 = J7,8 = 7.4 Hz,
=
1 H, PhCH2), 3.52–3.43 (m, 3 H), 2.79–2.72 (m, 1 H), 2.54–2.48
(m, 1 H), 2.35–2.20 (m, 1 H), 2.20–2.05 (m, 3 H), 2.05–1.95 (m, 2
2 H, fluorenyl 1-H and 8-H), 7.37 (pseudo t, 2 H, fluorenyl 3-H H), 1.93 (t, J = 2.6 Hz, 1 H, CϵCH), 1.78–1.70 (m, 2 H) ppm. 13
C
and 6-H), 7.28 (pseudo t, 2 H, fluorenyl 2-H and 7-H), 6.19 (br. s,
COOH), 5.46 (m, 1 H, NH), 4.49–4.33 (m, 3 H, CH2-O and α-H),
NMR (100 MHz, CDCl3): δ = 180.37 (O-C=O), 179.15 (N-C=O),
170.54 (C=N), 142.27, 133.67, 133.24, 132.16, 131.54, 129.71,
4.18 (t, J = 6.4 Hz, 1 H, fluorenyl 9-H), 3.24–3.21 (m, 2 H, ε-H2), 129.01, 128.89, 128.86, 127.63, 127.07, 126.38, 123.71, 120.71 (18ϫ
1.70–1.42 (m, 6 H, 3ϫ CH2) ppm. 13C NMR (100 MHz, CDCl3):
δ = 176.97 (COOH), 156.35 (CONH), 143.75, 143.60, and 141.28
Ar C), 83.47 (HCϵC), 70.27 (HCϵC), 69.99 (Pro C-α), 69.06
(PhCH2), 63.11 (C-COO), 57.05 (Pro C-δ), 34.53 (Pro C-β), 30.75
(fluorenyl C-4a, C-4b, C-8a, and C-9a), 127.74, 127.06, and 125.01 (CϵC-CH2-C), 24.36 [HCϵC-(CH2)2-C], 23.73 (Pro C-γ), 18.15
(fluorenyl C-2 to C-7), 120.00 (fluorenyl C-1 and C-8), 67.14 (CH2- (CϵC-C) ppm. C32H31N3NiO3 (564.30): calcd. C 68.11, H 5.54, N
O), 53.90 (C-α), 51.02 (C-ε), 47.07 (fluorenyl C-9), 31.68 (CH2), 7.45; found C 68.08, H 5.52, N 7.49.
28.31 (CH2), 22.55 (CH2) ppm. C21H22N4O4 (394.42): calcd. C
63.95, H 5.62, N 14.20; found C 64.01, H 5.58, N 14.23.
Alkylation of [NiII-(S)BPB-Gly] Complex with 6-Bromohex-1-yne
(14): Yield of 16: 68%. M.p. 87.5–88.5 °C; TLC: Rf(a & e) = 0.53
[CH2Cl2/Me2CO, 2:1]. [α]D = –1429 (c = 0.5, MeOH). ESI-MS:
calcd. for C33H33N3NiO3 [M + H]+: 578.19; found 578.4. 1H NMR
(400 MHz, CDCl3): δ = 8.12 (d, J = 8.4 Hz, 1 H, ArH), 8.04 (d, J
= 7.6 Hz, 2 H, ArH), 7.50–7.44 (m, 3 H, ArH), 7.33 (t, J = 7.6 Hz,
2 H, ArH), 7.26–7.10 (m, 3 H, ArH), 6.92 (d, J = 7.6 Hz, 1 H,
ArH), 6.66–6.60 (m, 2 H, ArH), 4.42 (d, J = 12.8 Hz, 1 H, PhCH2),
3.91 (dd, J1 = 3.4; J2 = 7.8 Hz, 1 H, Pro α-H), 3.57 (d, J = 12.8 Hz,
1 H, PhCH2), 3.52–3.43 (m, 3 H), 2.79–2.73 (m, 1 H), 2.56–2.46
(m, 1 H), 2.36–2.30 (m, 1 H), 2.16–2.02 (m, 4 H), 1.97–1.88 (m, 1
H), 1.91 (t, J = 2.4 Hz, 1 H, CϵCH), 1.76–1.61 (m, 2 H), 1.44–
1.32 (m, 2 H) ppm. 13C NMR (50 MHz, CDCl3): δ = 180.32 (O-
C=O), 179.30 (N-C=O), 170.46 (C=N), 142.27, 133.81, 133.21,
132.12, 131.54, 129.73, 128.94, 128.89, 128.85, 127.55, 127.14,
126.47, 123.66, 120.69 (18ϫ Ar), 83.86 (HCϵC), 70.28 (HCϵC),
Synthesis of (S)-2-(N-Benzylprolyl)aminobenzophenone (BPB) (11):
N-Benzyl--proline (prepared from proline as described pre-
viously)[24] (3.467 g, 16.9 mmol) was added at room temp. under N2
to a stirred freshly prepared transparent solution of PCl5 (7.035 g,
33.8 mmol) in anhydrous CH2Cl2 (55 mL). After 30 min, cold pe-
troleum ether was added and the acyl chloride precipitated as an
oil. The oil was dissolved in anhydrous CH2Cl2 (60 mL) under N2
and 2-aminobenzophenone (3.33 g, 16.9 mmol) was added in one
portion, followed by Et3N until pH 8. The mixture was stirred for
4 h at room temp., then washed with a saturated solution of
Na2CO3 and twice with H2O. The organic layer was dried with
anhydrous Na2SO4, filtered and the solvents evaporated under vac-
uum. The BPB was recrystallized from dried crude using EtOH.
Some product was also recovered from the EtOH washings. The
5312
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© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 5308–5314

Building Blocks for the Synthesis of “Clickable” Peptides
70.10 (Pro C-α), 68.83 (Bn CH2), 63.13 (C-COO), 56.99 (Pro C-δ),
34.53 (Pro C-β), 30.76 (HCϵC-CH2-C), 27.93 [HCϵC-(CH2)3-C],
24.30 (Pro C-γ), 23.67 [CϵC-(CH2)2-C], 18.14 (HCϵC-C) ppm.
C33H33N3NiO4 (578.33): calcd. C 68.53, H 5.75, N 7.27; found C
68.55, H 5.71, N 7.32.
Acknowledgments
We acknowledge financial support by the Italian Ministero dell’U-
niversità e della Ricerca (MUR) (PhD fellowship of A. L. C. I.)
and the Fondo per gli Investimenti della Ricerca di Base (FIRB)
(no. RBIN04TWKN; sponsoring of the cooperation between Uni-
versity of Florence and Harvard Medical School). We also thank
the Italian Fondazione Ente Cassa di Risparmio di Firenze for
financially supporting the Laboratory of Peptide and Protein
Chemistry and Biology of the University of Florence.
Hydrolysis of the Alkylated Complexes 15 and 16 and Fmoc Protec-
tion of the Free Amino Acids. General Procedure: A solution of the
alkylated complex (1.33 mmol) in MeOH (22.5 mL) was added to
warm 2  HCl (16 mL), the mixture was refluxed for 1 h. After
cooling to room temp., 1  NaOH was added until pH 6 and the
solvent was removed under vacuum. The solid residue was washed
with acetone, the dried solid product was dissolved in MeOH/H2O
(15:20 v/v) (70 mL) and then gently swirled ON with Chelex 100
resin in the H+ form. The mixture was filtered and the resin washed
with water, the combined filtrates were evaporated under vacuum,
and the residue lyophilized.
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in dioxane (15 mL) and then treated with 1  NaOH to obtain pH
8. The reaction mixture was stirred at room temp. for 4 h, followed
by the addition of water (7.5 mL) and acidification to pH 3 with
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MeOH in CH2Cl2 (0%-10%) to obtain the pure amino acid as a
yellow oil.
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2S-{[(9H-Fluoren-9-ylmethoxy)carbonyl]amino}-6-heptynoic
Acid
(19): Yield 24%. RP-UPLC: Rt = 1.49 min (50–100% of B in
3 min). [α]D = –3.0 (c = 1.0, MeOH). ESI-MS: calcd. for
1
C22H21NO4 [M + Na]+ 386.14; found 386.2. H NMR (400 MHz,
CDCl3): δ = 7.73 (d, J = 7.2 Hz, 2 H, fluorenyl 4-H and 5-H), 7.57
(d, J = 7.4 Hz, 2 H, fluorenyl 1-H and 8-H), 7.39 (pseudo t, 2 H,
fluorenyl 3-H and 6-H), 7.30 (pseudo t, 2 H, fluorenyl 2-H and 7-
H), 6.60 (br. s, COOH), 5.51 (m, 1 H, NH), 4.43–4.35 (m, 3 H,
CH2-O and α-H), 4.18 (t, J = 6.6 Hz, 1 H, fluorenyl 9-H), 2.08–
1.99 (m, 3 H), 1.94 (t, J = 2.4 Hz, 1 H, HCϵC), 1.80–1.75 (m, 1
H), 1.58–1.42 (m, 2 H, CH2) ppm. 13C NMR (100 MHz, CDCl3):
δ = 177.06 (COOH), 156.26 (CONH), 143.81, 143.62 and 141.27
(fluorenyl C-4a, C-4b, C-8a, and C-9a), 127.70, 127.06 and 125.05
(fluorenyl C-2 to C-7), 119.96 (fluorenyl C-1 and C-8), 83.49
(HCϵC), 69.11 (CH2-O), 67.06 (HCϵC), 53.77 (C-α), 47.11 (fluor-
enyl C-9), 31.33 (C-β), 24.28 (C-γ), 18.01 (C-δ) ppm. C22H21NO4
(363.41): calcd. C 72.71, H 5.82, N 3.85; found C 72.80, H 5.87, N
3.80.
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2S-{[(9H-Fluoren-9-ylmethoxy)carbonyl]amino}-7-octynoic
Acid
(20): Yield 35%. RP-UPLC: Rt = 1.49 min (50–100% of B in
3 min). [α]D = –3.1 (c = 1.0, MeOH). ESI-MS: calcd. for
1
C23H23NO4 [M + Na]+ 400.15; found 400.3. H NMR (400 MHz,
CDCl3): δ = 7.75 (pseudo d, J = 7.6 Hz, 2 H, fluorenyl 4-H and 5-
H), 7.59 (pseudo d, J = 7.6 Hz, 2 H, fluorenyl 1-H and 8-H), 7.37
(pseudo t, 2 H, fluorenyl 3-H and 6-H), 7.28 (pseudo t, 2 H, fluor-
enyl 3-H and 6-H), 5.79 (br. s, COOH), 5.48 (m, 1 H, NH), 4.44–
4.38 (m, 3 H, CH2-O and α-H), 4.21 (t, J = 6.8 Hz, 1 H, fluorenyl
9-H), 2.08–1.99 (m, 3 H), 1.94 (t, J = 2.4 Hz, 1 H, HCϵC), 1.80–
1.75 (m, 1 H), 1.58–1.42 (m, 4 H, 2ϫ CH2) ppm. 13C NMR
(100 MHz, CDCl3): δ = 176.63 (COOH), 156.17 (CONH), 143.83,
143.67 and 141.29 (fluorenyl C-4a, C-4b, C-8a, and C-9a), 127.71,
127.06, 125.04 (fluorenyl C-2 to C-7), 119.98 (fluorenyl C-1 and C-
8), 83.97 (HCϵC), 68.69 (CH2-O), 67.06 (HCϵC), 53.83 (C-α),
47.15 (fluorenyl C-9), 31.73 and 27.81 (C-β and δ), 24.31 (C-γ),
18.15 (C-ε) ppm. C23H23NO4 (377.43): calcd. C 73.19, H 6.14, N
3.71; found C 73.09, H 6.19, N 3.81.
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Eur. J. Org. Chem. 2008, 5308–5314
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5313

A. M. Papini et al.
FULL PAPER
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Received: July 18, 2008
[22] M. McLaughlin, R. M. Mohareb, H. Rapoport, J. Org. Chem.
2003, 68, 50–54.
Published Online: September 25, 2008
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Eur. J. Org. Chem. 2008, 5308–5314