Efficient synthesis of Fmoc-protected azido amino acids

Article abstract of DOI:10.1055/s-0030-1260950

The efficient two-step synthesis of Fmoc-protected L-azidoalanine and L-azidohomoalanine from readily available Fmoc-protected asparagine and glutamine, respectively, is reported. The synthetic route proceeds in good yield, requires no extra purification steps, and can be carried out on gram scale. The resulting azido amino acids are of sufficient purity for solid-phase peptide synthesis, as demonstrated in the synthesis of a model pentapeptide. Georg Thieme Verlag Stuttgart New York.

Full text of DOI:10.1055/s-0030-1260950

LETTER
1917
Efficient Synthesis of Fmoc-Protected Azido Amino Acids
Efficient
S
ynthesis
u
of Fmoc
A
zido A
H
A
cids eng Lau, David R. Spring*
Department of Chemistry, University of Cambridge, Lensfield Rd, Cambridge, CB2 1EW, UK
Fax +44(1223)336362; E-mail: spring@ch.cam.ac.uk
Received 15 April 2011
In our efforts to create azide-functionalized peptides on
solid phase using standard Fmoc chemistry, we looked to
develop an efficient synthesis for Fmoc-protected deriva-
tives of L-azidoalanine [Fmoc-Ala(N3)-OH, 1] and L-azi-
Abstract: The efficient two-step synthesis of Fmoc-protected L-
azidoalanine and L-azidohomoalanine from readily available Fmoc-
protected asparagine and glutamine, respectively, is reported. The
synthetic route proceeds in good yield, requires no extra purifica-
tion steps, and can be carried out on gram scale. The resulting azido dohomoalanine (Fmoc-Aha-OH, 2; Figure 1). As each
amino acids are of sufficient purity for solid-phase peptide synthe-
coupling step in solid-phase peptide synthesis requires
sis, as demonstrated in the synthesis of a model pentapeptide.
several equivalents of amino acid to ensure reaction com-
Key words: azides, amino acids, diazo transfer, Hofmann rear-
rangement, Fmoc solid-phase peptide synthesis
pletion, we aimed to find a route that would be short, high
yielding, and scalable, starting from simple and readily
available compounds.
There are several existing literature procedures for the
synthesis of Fmoc azido amino acids 1 and 2 (Table 1).
Fmoc-Aha-OH (2) has exclusively been synthesized from
the Boc-protected azido amino acid by removal of the Boc
protecting group and subsequent reprotection of the amine
with Fmoc.3,10 The analogous route to Fmoc-Ala(N3)-OH
(1) has also been reported.8 This synthetic strategy is re-
lated to the well-established methods for synthesizing un-
protected L-Aha via Boc-protected intermediates.11
Non-natural amino acids bearing azide groups are useful
building blocks in synthetic peptides and engineered pro-
teins, providing a bioorthogonal handle to which addition-
al functionality can be installed via Staudinger ligation1 or
Cu-catalyzed azide–alkyne cycloaddition reactions.2 A di-
verse range of applications has been developed for azido
amino acids based on these bioconjugation methods. For
example, Davis and coworkers have used L-azido-
homoalanine (Aha) as a site for attaching glycans onto a
LacZ reporter enzyme scaffold,3 achieving site-selective
glycosylation in a similar manner to post-translational
modification of proteins. The groups of Bertozzi and
Tirrell have exploited the ability of E. coli to substitute
Aha for methionine during translation,4 observing the pre-
sentation of azide groups on the cell surface of E. coli
cells.5
In search of a more direct and atom-efficient route, the nu-
cleophilic substitution of Fmoc-protected serine was ex-
plored, similar to the synthesis of the unprotected azido
amino acids reported by Roth and Thomas.11c,d However,
our attempts to obtain the desired azido amino acid from
Fmoc-Ser-OH by mesylation and substitution with sodi-
um azide were unsuccessful, consistent with the difficul-
ties previously reported by Panda and Rao when
attempting the reaction under either mesylation–substitu-
tion or Mitsunobu conditions.12
Azido amino acids such as L-azidoalanine [Ala(N3)] have
also been utilized in the synthesis of peptidomimetics, in-
cluding triazole-based macrocycles6 and antifreeze glyco-
peptides analogues7 via the Cu-catalyzed cycloaddition
reaction. Ala(N3) has also been incorporated into modi-
fied leucine zipper peptide sequences which gain tertiary
helical structure upon being constrained by macrocycliza-
tion with a bisalkyne functionalized linker.8 The proper-
ties of the azide group itself can also be utilized as an IR
probe to monitor protein folding and aggregation.9
Table 1 Summary of Literature Routesa
Synthetic approach
Boc replacement
Product Starting material Number of steps
(overall yield)
1
2
2
Boc-Dap-OH
homoserine
Boc-Dab-OH
3 (<50%)8
8 (<13%)10
4 (not reported)3
nucleophilic
substitution
1
Fmoc-Ser-OH
3 (63%)12,13
N3
N3
CO2H
diazo transfer
with triflyl azide
1
Fmoc-Dap-OH 1 (72–88%)6,7,9
FmocHN
CO2H
FmocHN
2
1
Hofmann/imidazole
diazo transfer
1
2
Fmoc-Asn-OH 2 (49–60%)b
Fmoc-Gln-OH 2 (46–55%)b
Figure 1 Fmoc-protected derivatives of L-azidoalanine [Fmoc-
Ala(N3)-OH, 1] and L-azidohomoalanine (Fmoc-Aha-OH, 2)
a Dap = 2,3-diaminopropionic acid; Dab = 2,4-diaminobutyric acid.
b This work.
SYNLETT 2011, No. 13, pp 1917–1919
x
x
.x
x
.2
0
1
1
Advanced online publication: 14.07.2011
DOI: 10.1055/s-0030-1260950; Art ID: D11811ST
© Georg Thieme Verlag Stuttgart · New York

1918
Y. H. Lau, D. R. Spring
LETTER
HN
NH2
Under these conditions, elimination to form the a,b-unsat-
urated compound is exclusively observed. Whilst mask-
ing the free carboxylate has been shown to resolve this
problem,12,13 it was decided not to introduce a new set of
protecting groups in our synthesis. Furthermore, the start-
ing material for accessing 2 via this route would be the
less available Fmoc-protected homoserine.
NH
N3
N3
O
O
O
O
H
H
N
N
NH2
N
N
N
H
H
H
O
O
Consequently, a copper-catalyzed diazo transfer strategy
from amine precursors was adopted, as diazo transfer us-
ing triflyl azide has previously been employed in the syn-
thesis of 1.6,7,9 However, to avoid safety issues associated
with using a potentially explosive diazo transfer reagent
on gram scale, it was decided to utilize imidazole-1-sulfo-
nyl azide to effect the diazo transfer in a safer and more
practical manner. This reagent can be synthesized on large
scale and is stable to long-term storage.14 It was also de-
cided to synthesize the amine precursors Fmoc-Dap-OH
(5) and Fmoc-Dab-OH (6) from Fmoc-protected aspar-
agine (3) and glutamine (4, Scheme 1). Both 3 and 4 are
standard, readily available protected amino acids that are
used in solid-phase peptide synthesis.
Figure 2 Model pentapeptide Ac-Ala(N3)-Ala-Arg-Ala-Aha-NH2
synthesized using Fmoc solid-phase peptide synthesis
In summary, the Hofmann rearrangement/diazo transfer
strategy is a simple and efficient route for synthesizing the
important protected amino acids Fmoc-Ala(N3)-OH (1)
and Fmoc-Aha-OH (2). Starting from readily available
materials, the procedure can easily be scaled up, and pure
products can be obtained without the need for column
chromatography or recrystallization. The ready availabil-
ity of these azido building blocks should have an impact
on numerous applications in chemical biology16 and
diversity-oriented synthesis.17
Fmoc-Asn-OH (3, 3.30 g, 9.31 mmol) was added to a solution of
[bis(trifluoroacetoxy)iodo]benzene (6.05 g, 14.1 mmol) in DMF–
H2O (2:1, 66 mL). After 15 min, pyridine (1.60 mL, 19.9 mmol) was
added, and the mixture was stirred at r.t. for 14 h. The solvent was
removed under reduced pressure, and the oily residue was dissolved
in H2O (50 mL). Concentrated HCl (1 mL) was added, and the acid-
ified solution was washed with Et2O (4 × 30 mL). The aqueous
phase was adjusted to pH 6 with 2 M NaOH solution, and the result-
ing precipitate was filtered, washed with H2O (5 × 30 mL), ice-cold
EtOH (10 mL), and Et2O (10 × 10 mL) and dried in vacuo to give
Fmoc-Dap-OH (5, 2.44g, 80%) as a beige powder. IR (ATR): 3297,
3037, 1724, 1686, 1631, 1575, 1533, 1491, 1448, 1409, 1324, 1248,
NH2
+
n
n
n
NH3
N3
O
b
a
–
FmocHN
CO2H
FmocHN
CO2
FmocHN
CO2H
3 (n = 1)
4 (n = 2)
5 (n = 1) 79–80%
6 (n = 2) 71–74%
1 (n = 1) 62–75%
2 (n = 2) 65–74%
Scheme 1 Reagents and conditions: a) [Bis(trifluoroacetoxy)io-
do]benzene, DMF–H2O (2:1), pyridine, 14 h; b) imidazole-1-sulfonyl
azide hydrochloride, K2CO3, H2O–MeOH–CH2Cl2 (1:1:1), pH 9, 18 h.
1
1068, 735 cm–1. H NMR (400 MHz, DMSO-d6): d = 7.89 (d,
Using [bis(trifluoroacetoxy)iodo]benzene and pyridine in
a Hofmann rearrangement based on literature proce-
dures,15 3 and 4 were converted to 5 and 6 in good yields
of 79–80% and 71–74%, respectively. This step can be
done on multigram scale, with the pure product easily iso-
lated by filtration.
J = 7.4 Hz, 2 H, ArH), 7.70 (d, J = 5.5 Hz, 2 H, ArH), 7.42 (t,
J = 7.2 Hz, 2 H, ArH), 7.33 (t, J = 7.0 Hz, 2 H, ArH), 6.78 (d,
J = 4.6 Hz, 1 H, CONH), 4.37–4.18 (m, 3 H, Fmoc-CH and CH2),
3.71 (m, 1 H, a-CH), 3.01 (dd, J = 10.6, 4.1 Hz, 1 H, one of b-H),
2.78 (t, J = 10.5 Hz, 1 H, one of b-H). HRMS (ES+): m/z [M + H]+
27
calcd for C18H19N2O4: 327.1345; found: 327.1348. [a]D +27.7 (c
0.41, DMSO).
From the amino compounds 5 and 6, initial attempts using
the original literature conditions14 for the diazo transfer
reagent with K2CO3 in MeOH resulted in significant
Fmoc deprotection due to the basic conditions of the reac-
tion. Changing to a biphasic solvent mixture of H2O,
MeOH, and CH2Cl2 adjusted to pH 9 with K2CO3 gave the
desired azido amino acids in yields of 62–75% and 65–
74% for 1 and 2, respectively.6 Notably, both compounds
were found to be >98% pure after workup, thus not requir-
ing column chromatography.
Fmoc-Dap-OH (5, 1.01 g, 3.11 mmol) was dissolved in a biphasic
mixture of H2O (15 mL), MeOH (30 mL), and CH2Cl2 (25 mL).
CuSO4·5H2O (5.0 mg, 0.020 mmol) and imidazole-1-sulfonyl azide
hydrochloride (2.02 g, 9.62 mmol) were added, and the mixture was
adjusted to pH 9 with aq K2CO3 solution. After stirring vigorously
for 18 h, the reaction mixture was diluted with CH2Cl2 (30 mL), and
the aqueous phase was isolated. The organic phase was extracted
with sat. NaHCO3 (2 × 50 mL). The combined aqueous extracts
were washed with Et2O (2 × 50 mL), acidified to pH 2 with concen-
trated HCl, and extracted with Et2O (3 × 60 mL). The organic ex-
tracts were dried over MgSO4 and concentrated in vacuo to give
Fmoc-Ala(N3)-OH (1, 820 mg, 75%) as a beige amorphous solid. IR
(ATR): 3303, 2103, 1690, 1536, 1446, 1341, 1259, 1088, 737 cm–1.
1H NMR (400 MHz, CDCl3): d = 7.76 (d, J = 7.5 Hz, 2 H, ArH),
7.59 (d, J = 6.2 Hz, 2 H, ArH), 7.40 (t, J = 7.5 Hz, 2 H, ArH), 7.31
(tt, J = 7.5, 1.1 Hz, 2 H, ArH), 5.65 (d, J = 7.6 Hz, 1 H, CONH),
4.57 (m, 1 H, a-CH), 4.49–4.37 (m, 2 H, Fmoc-CH2), 4.23 (t, J = 6.7
Hz, Fmoc-CH), 3.80 (m, 2 H, b-CH2). 13C NMR (100 MHz,
CDCl3): d = 173.3 (COOH), 156.2 (CONH), 143.6 (2 × CQAr),
141.4 (2 × CHAr), 127.9 (2 × CHAr), 127.2 (2 × CHAr), 125.1
(2 × CHAr), 120.1 (2 × CHAr), 67.6 (Fmoc-CH2), 53.9 (a-CH),
52.4 (b-CH2), 47.1 (Fmoc-CH). HRMS (ES+): m/z [M + H]+ calcd
To test the azido amino acids in Fmoc solid-phase peptide
synthesis, the pentapeptide Ac-Ala(N3)-Ala-Arg-Ala-
Aha-NH2 was synthesized on Rink amide resin (Figure 2).
The couplings proceeded smoothly with three equivalents
of each azido amino acid, and after cleavage from the res-
in, the crude product gave the correct mass by mass spec-
trometry and a single peak on HPLC (see Supporting
Information), with no significant byproducts or evidence
of diastereomers due to epimerization.
Synlett 2011, No. 13, 1917–1919 © Thieme Stuttgart · New York

LETTER
Efficient Synthesis of Fmoc Azido Amino Acids
1919
27
for C18H17N4O4: 353.1250; found: 341.1236. [a]D –10.3 (c 1.0,
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S. J.; Kirkpatrick, J.; Oldham, N. J.; Anthony, D. C.; Davis,
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Fmoc-Dab-OH (6) was prepared in a similar manner to 5 from
Fmoc-Gln-OH (2, 3.14 g, 8.52 mmol), [bis(trifluoroacetoxy)-
iodo]benzene (6.01 g, 14.0 mmol) and pyridine (1.60 mL, 19.9
mmol) to give the product (2.16 g, 74%) as a white powder. IR
(ATR): 2954, 1699, 1649, 1531, 1403, 1261, 1077, 760, 736 cm–1.
1H NMR (400 MHz, DMSO-d6): d = 7.89 (d, J = 7.4 Hz, 2 H, ArH),
7.67 (d, J = 7.1 Hz, 2 H, ArH), 7.42 (t, J = 7.4 Hz, 2 H, ArH), 7.33
(t, J = 7.3 Hz, 2 H, ArH), 6.63 (d, J = 3.4 Hz, 1 H, CONH), 4.34–
4.17 (m, 3 H, Fmoc-CH and CH2), 3.62 (m, 1 H, a-CH), 2.99–2.80
(m, 2 H, b-H), 1.95–1.84 (m, 1 H, one of g-H), 1.78–1.65 (m, 1 H,
one of g-H). HRMS (ES+): m/z [M + H]+ calcd for C19H21N2O4:
341.1501; found: 341.1492. [a]D27 +22.0 (c 1.0, DMSO).
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Fmoc-Aha-OH (2) was prepared in a similar manner to 1 from
Fmoc-Dab-OH (6, 1.03 g, 3.01 mmol), CuSO4·5H2O (5.0 mg, 0.020
mmol) and imidazole-1-sulfonyl azide hydrochloride (2.01 g, 9.59
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1
id. IR (ATR): 3329, 2106, 1693, 1535, 1445, 1242, 734 cm–1. H
NMR (400 MHz, CDCl3): d = 7.77 (d, J = 7.3 Hz, 2 H, ArH), 7.59
(d, J = 7.3 Hz, 2 H, ArH), 7.41 (t, J = 7.3 Hz, 2 H, ArH), 7.32 (tt,
J = 7.4, 1.1 Hz, 2 H, ArH), 5.41 (d, J = 7.0 Hz, 1 H, CONH), 4.56–
4.39 (m, 3 H, a-CH and Fmoc-CH2), 4.23 (t, J = 6.6 Hz, 1 H, Fmoc-
CH), 3.43 (m, 2 H, g-CH2), 2.19 (m, 1 H, one of b-CH2), 2.02 (m, 1
H, one of b-CH2). 13C NMR (100 MHz, CDCl3): d = 176.0 (COOH),
156.3 (CONH), 143.6 (2 × CQAr), 141.4 (2 × CQAr), 127.9
(2 × CHAr), 127.2 (2 × CHAr), 125.1 (2 × CHAr), 120.1
(2 × CHAr), 67.3 (Fmoc-CH2), 51.7 (a-CH), 47.7 (g-CH2), 47.2
(Fmoc-CH), 31.3 (b-CH2). HRMS (ES+): m/z [M + H]+ calcd for
27
C19H19N4O4: 367.1406; found: 367.1424. [a]D –14.4 (c 1.0,
MeOH, lit.10 [a]D –11.5).
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synlett.
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10.1038/ncomms1081 . For a recent application, see:
(b) Isidro-Llobet, A.; Murillo, T.; Bello, P.; Cilibrizzi, A.;
Hodgkinson, J. T.; Galloway, W. R. J. D.; Bender, A.;
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108, 6793.
Acknowledgment
We thank Cambridge Australia Scholarships and the Cambridge
Commonwealth Trust for the award of a scholarship to YHL. Also,
we thank the EPSRC, BBSRC, MRC, Wellcome Trust, and Francis
and Augustus Newman Foundation for funding.
References
(1) (a) Staudinger, H.; Meyer, J. Helv. Chim. Acta 1919, 2, 635.
(b) Saxon, E.; Bertozzi, C. Science 2000, 287, 2007.
Synlett 2011, No. 13, 1917–1919 © Thieme Stuttgart · New York