A concise route to L-azidoamino acids: L-azidoalanine, L-azidohomo-alanine and L-azidonorvaline

Article abstract of DOI:10.1055/S-0029-1218391

A simple and highly efficient synthetic route to three homologous azidoamino acids, starting from inexpensive, commercially available, protected natural amino acids is reported. The products can be used to introduce bioorthogonal handles into proteins. Ge

Full text of DOI:10.1055/S-0029-1218391

LETTER
607
A Concise Route to L-Azidoamino Acids: L-Azidoalanine, L-Azidohomo-
alanine and L-Azidonorvaline
A
Concise
Ro
t
L
e
-Azi doamin
f
o Aci dsanie Roth, Neil R. Thomas*
School of Chemistry, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK
Fax +44(115)9513564; E-mail: neil.thomas@nottingham.ac.uk
Received 1 October 2009
Dedicated to Prof. Gerry Pattenden on the occasion of his 70th birthday.
N3
N3
Abstract: A simple and highly efficient synthetic route to three ho-
mologous azidoamino acids, starting from inexpensive, commer-
cially available, protected natural amino acids is reported. The
products can be used to introduce bioorthogonal handles into pro-
teins.
N3
O–
O–
O–
H3+N
H3+N
H3+N
O
O
O
Key words: amino acids, azides, bioorganic chemistry, bioorthog-
onal chemistry, non-canonical
1
2
3
Figure 1 Three non-canonical azidoamino acids: L-azidoalanine
(1), L-azidohomoalanine (2) and L-azidonorvaline (3)
The creation of proteins that incorporate non-canonical
(unnatural) amino acids bearing side chains that can be se-
lectively modified has been the focus of increasing
amounts of research over the past decade. A number of
different methods have been devised including the use of
the amber suppression codon,1 the use of N-terminal mod-
ifying enzymes2 and of auxotrophic bacteria [typically E.
coli B834(DE3)] to introduce alkynyl,3 azide4 or ketone5
bearing amino acids that can be used as handles for further
modification via bio-orthogonal ‘click chemistry’.6 The
azido-group has proved to be the most versatile of these
because it can be used directly as an IR probe,7 it can se-
lectively undergo Huisgen [3+2] cycloadditions that can
be either copper(I)-catalysed (with an alkyne functiona-
lised probe),8 or metal-free (with a strained cyclooctyne
functionalised probe),9 and can react in a Staudinger reac-
tion with a phosphine-bearing probe.10 In a collaborative
project, the groups of Tirrell and Bertozzi were the first to
demonstrate that an azide in the form of L-azidohomo-
alanine (L-Aha, 2, Figure 1) could substitute for methion-
ine as a substrate for E. coli methionyl tRNA-synthetase
(MetRS) and be incorporated globally in place of me-
thionine in the E. coli outer-membrane protein OmpC
when expressed in auxotrophic E. coli.4 In this study it
was found that the MetRS adds L-Aha with a catalytic ef-
ficiency (kcat/KM) that was ~1/400th that of L-Met.
effective method of preparing these compounds on larger
scales (1–10 g). In this report we describe a generic and
robust route to L-azidoalanine (1), L-azidohomoalanine
(2), and L-azidonorvaline (3) from widely available, low
cost, protected a-amino acids. This route would also be
applicable to the synthesis of L-azidonorleucine from
commercially available N-Boc-L-norleucine with an addi-
tional esterification step. There have been a number of
previous syntheses of L-azidoalanine (1),12 L-azido-
homoalanine (2)13,14,15 and L-azidonorvaline (3)16 that
give both the protected and fully deprotected amino acids.
Of those that give the deprotected azidoamino acids, a
number suffer from disadvantages such as starting materi-
als that are either expensive or not readily available, the
required use of hazardous reagents, or involve either a
large number of transformations, or have a poor overall
yield.
Tirell and co-workers13,14 have recently reported two syn-
theses for L-Aha (2) – the first being a modified version of
Mangold’s original synthesis.15 Although starting from a
readily available amino acid derivative (Boc-L-homo-
serine), this protocol uses a rather complicated protecting
group strategy that requires the use of diazomethane, a
hazardous reagent that requires specific handling and
glassware. The alternative, improved synthesis they de-
scribed avoids this step but starts from expensive Boc-
Dab (Boc-diaminobutyric acid) and employs triflic azide,
a relatively unstable reagent that has to be freshly pre-
pared prior to use. For the preparation of larger quantities
of proteins incorporating L-azidohomoalanine (2), Tirrell
et al. have used a-amino-g-butyrolactone17 to give rac-
azidohomoalanine in reasonable quantities.15 Whilst only
the L-enantiomer was incorporated into the proteins of in-
terest, the effect of the D-enantiomer on cell growth and
protein production was not reported, but could potentially
be detrimental.
Later it was shown that other L-azidoamino acids such as:
L-azidoalanine (1), L-azidonorvaline (3) (Figure 1) and L-
azidonorleucine (not shown), could be incorporated into
OmpC, albeit with much lower efficiency than L-Aha
(2).11 The development of an increasing variety of meth-
ods with which to incorporate azidoamino acids such as 2
into proteins has generated the need for a facile and cost
SYNLETT 2010, No. 4, pp 0607–0609
0
1.
0
3.
2
0
1
0
Advanced online publication: 27.11.2009
DOI: 10.1055/s-0029-1218391; Art ID: D28009ST
© Georg Thieme Verlag Stuttgart · New York

608
S. Roth, N. R. Thomas
LETTER
We proposed that the three homologous L-azidoamino >99% by either recrystallisation from ethanol/water or
acids 1, 2 and 3 could be prepared starting from inexpen- ion-exchange chromatography.5
sive, commercially available, protected amino acids em-
ploying a common synthetic scheme that avoids handling
highly dangerous or toxic compounds such as diaz-
omethane, or the use of the unstable triflic azide.
After establishing a viable route for L-Aha (2), we then
turned our attention to the synthesis of the homologues 1
and 3 as single enantiomers. Applying the same strategy
starting from either N-Boc- and O-benzyl-protected L-
Initially, we developed the synthetic route shown in serine 9 or protected L-glutamic acid 8, which are both
Scheme 1 for the synthesis of L-Aha (2) as this has been low cost, commercially available compounds, the expect-
shown to be by far the most translationally active me- ed products 1 and 3 could be obtained in high overall
thionine surrogate4,5 and, consequently, the azidoamino yields of 68% (over three steps) and 62% (over four
acid most widely employed. Starting from N-Boc- and O- steps), respectively (Scheme 2).
benzyl-protected L-aspartic acid 4, the g-carboxylic acid
was reduced directly via the mixed anhydride formed with
1 equiv NMM
1 equiv i-BuOCOCl
6 equiv NaBH4
THF–MeOH (1:2)
–10 °C→r.t., 1 h
O
OH
isobutyl chloroformate.18 This step was carried out on a
multigram scale to give 5 reproducibly in quantitative
yield and further purification was not required. The result-
ing alcohol 5 was then subjected to a standard mesylation
reaction using mesylchloride and triethylamine to give
mesylate 6.19 The mesylate function was then displaced
with an azide group by treatment with sodium azide in
DMF at slightly elevated temperature to give protected
azide 7 in an excellent yield of 92%.20 An initial attempt
to deprotect the amino acid in two subsequent steps using
an acid-mediated deprotection of the Boc group followed
by a basic cleavage of the benzyl ester did result in the free
amino acid, however, due to the additional purification
step, the overall yield was reduced.
OH
n
BocHN
CO2Bn
9, n = 0
10, n = 2
1.2 equiv MsCl
n = 2: 95%
BocHN
CO2Bn
8
2.4 equiv Et3N
CH2Cl2
0 °C→r.t., 15 min
n = 0: 95%
n = 2: 81%
1.5 equiv NaN3,
DMF,
40 °C, 3–3.5 h,
N3
OMs
n
n
n = 0: 73%,
n = 2: 81%
BocHN
CO2Bn
BocHN
CO2Bn
13, n = 0
14, n = 2
11, n = 0
12, n = 2
5 equiv BBr3
CH2Cl2
–10 °C→r.t., 3 h
n = 0: 98%
n = 2: quant
1 equiv NMM
1 equiv i-BUOCOCl
6 equiv NaBH4
O
THF–MeOH (1:2)
–10 °C→r.t., 0.5 h
N3
n
OH
OH
quant
–
H3+N
CO2
BocHN
CO2Bn
BocHN
CO2Bn
1, n = 0
4
5
3, n = 2
1.2 equvi MsCl
2.4 equiv Et3N
CH2Cl2
Scheme 2 Synthesis of L-azidoalanine (1) and L-azidonorvaline (3)
0 °C→r.t.
15 min
93%
It is noteworthy that, after reduction of the protected
glutamic acid 8, purification of the alcohol 10 by column
chromatography was necessary, presumably to remove
traces of boron-based compounds and any aldehyde
present. If the alcohol is subjected to the mesylation reac-
tion without prior purification, the yield of the mesylation
reaction decreases from 81% to 42%.
1.5 equiv NaN3
DMF
OMs
N3
40 °C, 4 h
92%
BocHN
CO2Bn
BocHN
CO2Bn
7
6
5 equiv BBr3
CH2Cl2
–10 °C→r.t., 3 h
quant
In conclusion, a facile and robust synthesis of the three ho-
mologous azidoamino acids: L-azidoalanine (1), L-azido-
homoalanine (2) and L-azidonorvaline (3), from
inexpensive, commercially available starting materials
has been developed. The synthetic route is high-yielding,
applicable to all three compounds and could also be ap-
plied to the synthesis of L-azidonorleucine without modi-
fication. This route does not require highly hazardous or
unstable reagents and so could be performed on multi-
gram scales when large quantities of azidoamino acid
bearing protein were required for further selective modifi-
cation. The introduction of L-Aha (2) into proteins also al-
lows mild fragmentation of peptides and proteins at
specific positions.
N3
–
H3+N
CO2
2
Scheme 1 Synthesis of L-azidohomoalanine (L-Aha, 2)
We therefore applied a global deprotection strategy em-
ploying boron tribromide21 that afforded 2 in quantitative
yield after crystallisation; an overall yield of 86% from
4.22 The purity of the material obtained was ~98% (the
major by-product resulting from a displacement of the
azide group with a bromide), which could be enhanced to
Synlett 2010, No. 4, 607–609 © Thieme Stuttgart · New York

LETTER
A Concise Route to L-Azidoamino Acids
609
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Acknowledgment
This work was funded by the BBSRC SCIBS initiative (Project
grant BBE0140891) to N.R.T.
(19) To a solution of alcohol 5 (269 mg, 0.87 mmol, 1.0 equiv)
in CH2Cl2 (5 mL) at 0 °C, was added Et3N (288 mL,
References and Notes
2.09 mmol, 2.4 equiv) followed by dropwise addition of
methylsulfonyl chloride (81 mL, 1.04 mmol, 1.2 equiv). The
ice-bath was removed and the solution was stirred at r.t. for
15 min. Sat. NaHCO3 (5 mL) was added and the layers were
separated. The organic layer was washed with brine (2 ×
5 mL) and the combined aqueous layer was back-extracted
with CH2Cl2 (2 × 10 mL). The combined organic layer was
dried over MgSO4, the solids filtered off and the solvent
removed in vacuo. The crude product was purified by
column chromatography (silica; PE–EtOAc, 7:3) to give a
colourless solid (312 mg, 0.81 mmol, 93%); mp 61–63 C
(CHCl3); [a]D28 –37.1 (c 0.11, CHCl3); IR: 3009, 1711, 1500,
1364, 1175 cm–1; 1H NMR (400 MHz, CDCl3): d = 1.39 (s,
9 H, tBu), 2.08 (m, 1 H, Hb1), 2.29 (m, 1 H, Hb2), 2.92 (s,
3 H, SO2CH3), 4.24 (m, 2 H, Hg), 4.43 (m, 1 H, Ha), 5.15 (s,
2 H, CH2Ph), 5.26 (bd, J = 7.0 Hz, 1 H, NH), 7.33 (m, 5 H,
Ar); 13C NMR (100 MHz, CDCl3): d = 28.2 [C(CH3)3], 31.8
(Cb), 37.1 (SO2CH3), 50.4 (Ca), 65.8 (Cg), 67.5 (CH2Ph),
80.3 [C(CH3)3], 128.4, 128.5, 128.7, 135.0 (Ar), 155.3 (t-
BuOCONHR), 177.5 (CO2Bn); MS (ESI+): m/z [M + Na]+
calcd for C17H25NNaO7S: 410.1244; found: 410.1225.
(20) To a solution of mesylate 6 (222 mg, 0.56 mmol, 1.0 equiv)
in anhydrous DMF (2 mL), was added NaN3 (54 mg,
0.84 mmol, 1.5 equiv) in one portion. The suspension was
stirred at 40 °C for 4 h, then the solvent was removed in
vacuo and the crude product was purified by column
chromatography (silica; PE–EtOAc, 4:1). The azide 7 was
obtained as a colourless oil (171 mg, 0.51 mmol, 92%);
[a]D28 +2.8 (c 0.71, CHCl3); IR: 3434, 2981, 2104, 1712,
1499, 1160 cm–1; 1H NMR (400 MHz, CDCl3): d = 1.41 (s,
9 H, tBu), 1.89 (m, 1 H, Hb1), 2.08 (m, 1 H, Hb2), 3.34 (t, J =
6.7 Hz, 2 H, Hg), 4.41 (m, 1 H, Ha), 5.13 (d, J = 12.3 Hz, 1 H,
CH2Ph), 5.18 (d, J = 12.3 Hz, 1 H, CH2Ph), 5.19 (br s, 1 H,
NH), 7.34 (m, 5 H, Ar); 13C NMR (100 MHz, CDCl3): d =
28.2 [C(CH3)3], 31.7 (Cb), 47.6 (Cg), 51.5 (Ca), 67.3
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Synlett 2010, No. 4, 607–609 © Thieme Stuttgart · New York