Improved solid-phase peptide synthesis method utilizing alpha-azide-protected amino acids.

Article abstract of DOI:10.1021/ol0155485

[structure: see text]. Pure alpha-azido acids were prepared using an efficient diazo transfer method followed by buffered workup. These building blocks were used to prepare small peptides on Wang resin by two approaches. Peptides prone to diketopiperazine formation were prepared in good yields by coupling acids to resin bound iminophosphoranes during Fmoc-Wang synthesis. The iminophosphoranes can also be hydrolyzed under neutral conditions to provide unprotected amines ready for further coupling.

Full text of DOI:10.1021/ol0155485

ORGANIC
LETTERS
2001
Vol. 3, No. 5
781-783
Improved Solid-Phase Peptide Synthesis
Method Utilizing r-Azide-Protected
Amino Acids
Joseph T. Lundquist, IV and Jeffrey C. Pelletier*
DiVision of DiscoVery Chemistry, Wyeth-Ayerst Research, 145 King of Prussia Road,
Radnor, PennsylVania 19087
Pelletj@WAR.Wyeth.com
Received January 11, 2001
ABSTRACT
Pure r-azido acids were prepared using an efficient diazo transfer method followed by buffered workup. These building blocks were used to
prepare small peptides on Wang resin by two approaches. Peptides prone to diketopiperazine formation were prepared in good yields by
coupling acids to resin bound iminophosphoranes during Fmoc-Wang synthesis. The iminophosphoranes can also be hydrolyzed under neutral
conditions to provide unprotected amines ready for further coupling.
Small peptides constitute a useful class of bioactive mol-
ecules despite efforts to find nonpeptide substrates for
biological targets. In fact, analysis of the Physicians’ Desk
Reference reveals that there are currently several small
peptides used for therapeutic indications in which no
nonpeptide alternative treatments are available. While re-
search quantities of larger peptides are generally obtained
from natural sources or recombinant techniques, organic
synthesis is the method of choice for smaller peptides and
analogues containing nonnatural elements.1
Merrifield introduced solid-phase peptide synthesis (SPPS)
in 1963 utilizing acid-labile protecting groups as well as a
resin-substrate linker that was cleaved under strongly acidic
conditions.2 Similar work based on the 9-(fluorenylmethoxy)-
carbonyl (Fmoc) protecting group in conjunction with the
Wang linker has expanded the operation of peptide synthesis
into the typical organic chemistry lab due to the mild
conditions employed.3 Although useful, lack of orthogonal
protecting groups for resin-bound side chain manipulation
and diketopiperazine (DKP) formation still present significant
challenges for Fmoc-Wang-based SPPS.4 A strategy to
prevent DKP formation is known but not readily imple-
mented due to the need for hindered linker preparation and
N-protected amino acid chlorides.5
The use of azides as amine protecting groups in SPPS has
been described.6 The method is particularly useful for
hindered couplings; however, the need for chromatographic
purification of azido acids and deprotections involving mild
heat, thiols, and tertiary amines present drawbacks.
Solution-phase amide bond formation from carboxylic
acids and iminophosphoranes (prepared from the correspond-
ing azides and phosphines) is well-known.7 Although the
condensation typically takes place at elevated temperatures,
similar transformations have been shown to occur at room
temperature with activated acids.8 Modifications of this
coupling procedure have recently been applied to solid-phase
organic synthesis (SPOS)9 as well as amide-forming ligation
reactions.10 Our goal was to combine and successfully modify
these protocols for application toward key steps in Fmoc-
Wang SPPS as well as improving methods utilizing a-azido
acids as building blocks.
(4) (a) Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35,
161-214. (b) Pedroso, E.; Grandas, A.; de las Heras, X.; Eritja, R.; Giralt,
E. Tetrahedron Lett. 1986, 27, 743-746. (c) Barany, G.; Albericio, F. J.
Am. Chem. Soc. 1985, 107, 4936-4942. (d) Hardy, P. M.; Sheppard, P.
W. J. Chem. Soc., Perkin Trans. 1 1983, 723-729.
(5) Akaji, K.; Kiso, Y.; Carpino, L. A. J. Chem. Soc., Chem Commun.
1990, 584-586.
(6) (a) Meldal, M.; Juliano, M. A.; Jansson, A. M. Tetrahedron Lett.
1997, 38, 2531-2534. (b) Tornoe, C. W.; Davis, P.; Porreca, F.; Meldal,
M. J. Pept. Sci. 2000, 6, 594-602.
(1) (a) Dutta, A. S. In Amino Acids, Peptides and Proteins; Davies, J.
S., Ed.; Royal Society of Chemistry: Cambridge, U.K., 1999; Vol. 30,
Chapter 3, pp 163-284. (b) Mayo, K. H. Tibtech 2000, 18, 212-217.
(2) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149-2154.
(3) (a) Wang S. S. J. Am. Chem. Soc. 1973, 95, 1328-1333. (b) Carpino,
L. A.; Han, G. Y. J. Org. Chem. 1972, 37, 3404-3409.
10.1021/ol0155485 CCC: $20.00 © 2001 American Chemical Society
Published on Web 02/14/2001

Table 1. R-Azido Acids Prepared in Scheme 1
a
a
entry
azido acid
[R]25
yield
entry
azido acid
N3-Gln
N3-Asp(t-Bu)
N3-Glu(Bzl)
N3-Ser(t-Bu)
N3-D-Ser(t-Bu)
N3-Thr(t-Bu)
N3-Met
[R]25
yield
D
D
1
2
3
4
5
6
7
8
9
10
11
12
13
14
N3-Gly
N3-Phe
N3-D-Phe
N3-Val
N3-D-Val
N3-Ala
-
68f
62
68
75
80
66
68
89
73
76
80
85
87
48
15
16
17
18
19
20
21
22
23
24
25
26
27
28
-73.3, MeOH
-65.9
49
82
42
84
84
80
86
75
86
41
70
46
66
86
-74.2b
+64c
-46.8
+29.3
-27.4
+31.6
-47.8
+50.8
+22.1
-13.0
-33.0
+38.3
+108.6
-69.6
+145.9d
-163.6e
N3-Leu
N3-Ile
-98.5
N3-IBA
-
N3-D-Ile
N3-D-allo-Ile
N3-tert-Leu
N3-Phg
N3-D-Phg
N3-Asn
N3-Trp(t-Boc)
N3-Cys(4-MeO-Bzl)
N3-Tyr(t-Bu)
N3-Arg(Mtr)
N3-Lys(t-Boc)
N3-His
+31.0
-53.0
-40.7
-19.5, MeOH
-19.0
-102.1, MeOH
-98.0, MeOH
a c ) 1.0, CHCl3 or MeOH if specified. b Lit.16 [R]25 ) -67.9 (c ) 107, CHCl3). c Lit.16 [R]D ) +68.6 (c ) 1.4, CHCl3). d Lit.16 [R]25 ) +175 (c )
D
D
f
1.06, CHCl3). e Lit.16 [R]D ) -169 (c ) 1.4, CHCl3). Synthesized trough nucleophilic substitution of bromoacetic acid by sodium azide.17 Phenylglycine
) Phg; Isobutyric Acid ) IBA; 4-methoxybenzyl ) 4-MeO-Bzl; 4-methoxy-2,3,6-trimethylbenzenesulfonyl ) Mtr.
Successful implementation required readily available a-
azido acids in chiral form (Table 1, 1-28). We chose the
copper(II)-catalyzed diazo transfer method of Wong11 (Scheme
1) to prepare 1-28,6b,12 since other methods were less general
To prove useful to the field of SPPS, an ideal coupling
method required good yields of products with little or no
racemization. Preparation of peptides with the Glycine-
Proline (GlyPro) C-terminus was reported to be unsuccessful
due to DKP formation.4a Hence, Wang resin-bound FmocPro
was deprotected, and the product was coupled to R-azido
glycine (1) under standard conditions. This resin-bound azide
was then treated with trimethylphosphine in dry dioxane and
commercially available Fmoc-protected glycine O-succin-
imide ester. Cleavage from the support and purification by
reversed-phase HPLC provided the desired FmocGlyGlyPro
(29) in 70% yield (Scheme 2, path A). When the preparation
of this compound was attempted with standard Fmoc-Wang
chemistry, no product was detected (see Table 2, entry 29).
Other DKP-susceptible peptides4,5 were also prepared using
both methods. Substantial yield improvements were noted
Scheme 1 R-Azido Acid Synthesisa
a Reagents and conditions: (i) Tf2O, NaN3; (ii) CuSO4, K2CO3,
H2O, MeOH, CH2Cl2.
or involved cumbersome workups.6,7c,13 Buffered extraction
of the trifluoromethanesulfonamide byproduct during workup
provided analytically pure azido acids in most cases without
chromatography. Protection of the R-carboxylate was not
required. Epimerization was negligible, and the method
worked equally well on hindered substrates or amino acids
possessing various side chain protecting groups (16-20 and
23-27). Additionally, substrates with unprotected nitrogen
containing side chains were readily converted to R-azido
acids (14, 15, and 28).
(11) Alper, P. B.; Hung, S.-C.; Wong, C.-H. Tetrahedron Lett. 1996,
37, 6029-6032.
(12) Representative Azido Acid Synthesis by Diazo Transfer. Synthesis
of Azido-Ile (8). The diazo transfer reactions utilize the method of Wong11
for carbohydrates with an customized workup to accommodate the free acid
products. Triflyl azide preparation: A solution of sodium azide (1.78 g,
27.45 mmol) was dissolved in distilled H2O (4.5 mL) with CH2Cl2 (7.5
mL) and cooled on an ice bath. Triflyl anhydride (0.93 mL, 5.55 mmol)
was added slowly over 5 min while stirring continued for 2 h. The mixture
was placed in a separatory funnel and the CH2Cl2 phase removed. The
aqueous portion was extracted with CH2Cl2 (2 × 3.75 mL). The organic
fractions, containing the triflyl azide, were pooled and washed once with
saturated Na2CO3 and used without further purification. L-Ile (366 mg, 2.79
mmol) was combined with K2CO3 (577.5 mg, 4.19 mmol), CuIISO4
pentahydrate (6.98 mg, 27.9 µmol), distilled H2O (9 mL), and CH3OH (18
mL). The triflyl azide in CH2Cl2 (15 mL) was added, and the mixture was
stirred at ambient temperature overnight. Subsequently, the organic solvents
were removed under reduced pressure, and the aqueous slurry was diluted
with H2O (50 mL). This was acidified to pH 6 with concentrated HCl and
diluted with 0.25 M, pH 6.2 phosphate buffer (50 mL) and extracted with
EtOAc (4×) to remove sulfonamide byproduct. The aqueous phase was
then acidified to pH 2 with concentrated HCl. The product was obtained
from EtOAc extractions (3×). The organic extracts were combined, dried
(MgSO4), and evaporated to dryness giving 390 mg of the pale oil (8) in
(7) (a) Garcia, J.; Urp´ı, F.; Vilarrasa, J. Tetrahedron Lett. 1984, 25,
4841-4844. (b) Zaloom, J.; Calandra, M.; Roberts, D. C. J. Org. Chem.
1985, 50, 2601-2603. (c) Hoffman, R. V.; Kim, H.-O. Tetrahedron 1992,
48, 3007-3020. (d) Ghosh, S. K.; Singh, U.; Mamdapur, V. R. Tetrahedron
Lett. 1992, 33, 805-808. (e) Urp´ı, F.; Vilarrasa, J. Tetrahedron Lett. 1986,
27, 4623-4624. (f) Murahashi, S.-I.; Taniguchi, Y.; Imada, Y.; Tanigawa,
Y. J. Org. Chem. 1989, 54, 3292-3303. (g) Stra¨ssler, C.; Heimgartner, H.
HelV. Chim. Acta 1997, 80, 2058-2065. (h) Mizuno, M.; Muramoto, I.;
Kobayashi, K.; Yaginuma, H.; Inazu, T. Synthesis 1999, 162-165. (i)
Sikora, D.; Gajda, T. Tetrahedron 2000, 56, 3755-3761.
(8) Bosch, I.; Romea, P.; Urp´ı, F.; Vilarrasa, J. Tetrahedron Lett. 1993,
34, 4671-4674.
(9) (a) Tang, Z.; Pelletier, J. C. Tetrahedron Lett. 1998, 39, 4773-4776.
(b) Malkinson, J. P.; Falconer, R. A.; Toth, I. J. Org. Chem. 2000, 65,
5249-5252.
89% yield with no need for further purification. [R]25 ) -33.0 (c ) 1.0
D
in CHCl3); 1H NMR (300 MHz, CDCl3) δ 3.84 (d, J ) 5.8 Hz, 1H), 2.15-
1.95 (m, 2H), 1.70-1.55 (m, 1H), 1.45-1.25 (m, 1H), 1.04 (d, J ) 6.8
Hz, 3H), 0.94 (t, J ) 7.4 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 174.5,
66.9, 37.2, 24.9, 15.9, 11.5. Anal. Calcd for C6H11N3O2: C, 45.85; H, 7.05;
N, 26.74. Found: C, 46.19; H, 7.29; N, 26.72.
(10) (a) Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett. 2000,
3, 9-12. (b) Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett. 2000,
2, 1939-1941. (c) Saxon, E.; Armstrong, J. I.; Bertozzi, C. R. Org. Lett.
2000, 2, 2141-2143. (d) Saxon, E.; Bertozzi, C. R. Science 2000, 287,
2007-2010.
(13) Zaloom, J.; Roberts, D. C. J. Org. Chem. 1981, 46, 5173-5176.
782
Org. Lett., Vol. 3, No. 5, 2001

of crude tripeptides constructed with hindered isoleucine and
D-allo-isoleucine in both the second and third positions
indicated homogeneous products (Table 2, entries 33-36).
The alpha protons of the isoleucyl and D-allo-isoleucyl
subunits in each case have unique signals coupled to the
chiral â-proton. Hence, any racemization of the D-allo-
isoleucyl subunit during the preparation of 33 would lead to
its contamination with tripeptide 34. The opposite would be
true for the preparation of 34. No cross contamination could
Scheme 2. Peptide Preparation from R-Azido Acidsa
1
be observed by 300 MHz H NMR in either case. Similar
rationale was applied to tripeptides 35 and 36 with the same
results as above. The method confirmed that the stereochem-
ical integrity of the amino acid components surrounding the
newly formed amide bond was not compromised while
coupling with the iminophosphorane.
To expand the general utility of this methodology, N-
terminal azidotetrapeptides were synthesized using azido
acids as building blocks (Scheme 2, path B). Azides are
readily converted to amines under mildly reductive condi-
tions, employing phosphines in aqueous solvent systems.15
Furthermore, the transformation proceeds under neutrality
and avoids acid/base-catalyzed side reactions. A number of
N-terminal azidotetrapeptides were prepared using standard
coupling methods followed by the mild azide reduction
technique described above (Scheme 2, path B and Table 2,
entries 37-44). The protocol suggests that a neutral, mild
deprotection of resin-bound azides could add additional
orthogonality to SPPS. Good yields were obtained in most
cases except when hindered amino acid couplings were
involved. The success of this method indicates that azido
acids prepared by a more elaborate, asymmetric synthesis16
could be incorporated directly into the peptides. This
alleviates the need for reduction and then reprotection of the
R-amino group of these valuable residues prior to coupling.
The methods described in this work show the robust and
versatile nature of chiral R-azido acids as peptide building
blocks. Increased yields for DKP susceptible peptides,
flexibility in coupling methods, neutral reaction conditions,
and the ability to complement standard Fmoc-Wang chem-
istry make the azido acid coupling strategy detailed here a
useful addition to organic synthesis methodology.
a Reagents and conditions: (i) 20% piperidine, DMF, 2 min.,
then 8 min.; (ii) azido acid, DCC, HOBT, (4.0 equiv each), DMF;
(iii) trimethylphosphine (1.5 equiv), FmocAAOSu (4.0 equiv),
dioxane; (iv) TFA-DCM(1:1); (v) trimethylphosphine (6.0 equiv),
aq dioxane.
with the azide coupling procedure (Table 2, entries 30-32).
We attribute the increase in yields from the azido acid
coupling method to suppresion of DKP formation since a
closely related protocol was shown to inhibit the formation
of δ-lactams.9a Although the reaction proceeds via an
iminophosphorane intermediate, progress can be followed
using the well-adopted Kaiser test.14 Epimerization does not
appear to be a significant problem since 1H NMR inspection
Table 2. Peptides Prepared from R-Azidoacids
entry
sequence
yielda,b
epimerization
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
FmocGlyGlyPro
FmocGlyThrPro
FmocGly-D-ValPro
FmocValTyrPro
Gly-D-allo-IlePhe
GlyIlePhe
70 (0)
81 (24)
46 (0)
71 (0)
-
-
-
-
76
20
15
43
53
21
46
66
-
-
-
-
Acknowledgment. We thank Wyeth-Ayerst Research for
postdoctoral funding for J.T.L. and Jay Wrobel for helpful
discussions.
<1%
<1%
<1%
<1%
-
-
-
-
-
-
-
-
Supporting Information Available: Experimental pro-
cedures and analytical data for all azido acids (1-28) and
peptides (29-44). This material is available free of charge
via the Internet at http://pubs.acs.org.
D-allo-IleGlyGly
IleGlyGly
N3AspTyrLeuVal
N3-D-Val-t-LeuTyrAla
N3IleAibThrPhe
N3-t-LeuVal-D-PheLeu
N3LeuGlyPheVal
N3MetAlaValPhe
N3Ala-D-IleAlaPhe
N3-t-LeuGlyIlePhe
OL0155485
(14) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 595-598.
(15) Vaultier, M.; Knouzi, N.; Carrie, R. Tetrahedron Lett. 1983, 24,
763-764.
a Yields are for purified products. b Yields in parentheses are for products
prepared using the Fmoc-Wang SPPS method.
(16) Evans, D. A.; Britton, T. C.; Ellman, J. A.; Dorow, R. L. J. Am.
Chem. Soc. 1990, 112, 4011-4030.
(17) Alvarez, S. G.; Alvarez, M. T. Synthesis 1997, 413-414.
Org. Lett., Vol. 3, No. 5, 2001
783