Benzotriazole-assisted solid-phase assembly of Leu-Enkephalin, amyloid β segment 34-42, and other "difficult" peptide sequences

Article abstract of DOI:10.1021/jo8026214

Microwave-assisted solid-phase syntheses of six "difficult" peptides, H-VVSVV-NH₂ (3), H-VVVSVV-NH₂(4), H-VIVIG-OH (5), H-TVTVTV-NH₂ (6), H-VKDGYI-NH₂ (7), and H-VKDVYI-NH₂ (8), were achieved utilizing N-(Fmoc-α-aminoacyl) benzotriazoles. Extension to the syntheses of Leu-enkephalin (9) and amyloid-β (34-42) (10) demonstrates that this strategy comprises an efficient route to new and known "difficult" peptides.

Full text of DOI:10.1021/jo8026214

Benzotriazole-Assisted Solid-Phase Assembly of Leu-Enkephalin,
Amyloid ꢀ segment 34-42, and other “Difficult” Peptide Sequences
Alan R. Katritzky,*,‡ Danniebelle N. Haase,‡ Jodie V. Johnson,§ and Alfred Chung|
Center for Heterocyclic Compounds and Department of Chemistry, UniVersity of Florida, GainesVille,
Florida 32611-7200, and PROTEOMICS, Interdisciplinary Center for Biotechnology Research (ICBR),
UniVersity of Florida, GainesVille, Florida 32611-1376
katritzky@chem.ufl.edu
ReceiVed NoVember 26, 2008
Microwave-assisted solid-phase syntheses of six “difficult” peptides, H-VVSVV-NH2 (3), H-VVVSVV-
NH2 (4), H-VIVIG-OH (5), H-TVTVTV-NH2 (6), H-VKDGYI-NH2 (7), and H-VKDVYI-NH2 (8), were
achieved utilizing N-(Fmoc-R-aminoacyl)benzotriazoles. Extension to the syntheses of Leu-enkephalin
(9) and amyloid-ꢀ (34-42) (10) demonstrates that this strategy comprises an efficient route to new and
known “difficult” peptides.
Introduction
aminoacylation and/or deprotection reactions at various stages
3a-d
in the synthetic scheme
have resulted in low yields and
Solution-phase and especially the solid-phase peptide syn-
thesis (SPPS) of potentially bioactive peptides is of great
interest.1a,b While SPPS has enabled major advances in scope,
yields, purities, and reaction times,1a,c SPPS2a,b has also
encountered “difficult” peptide sequences when incomplete
purities.1a Such difficulties can arise in attempting to form a
peptide link (i) due to steric effects when both amino acid units
possess ꢀ-branched side chains (e.g., valine, isoleucine and
threonine)3b and (ii) as a result of the formation of secondary
structures by intra- and interchain hydrogen-bonded associa-
tions.3a-d In such cases, the synthesized peptides can be partly
‡ Center for Heterocyclic Compounds, Department of Chemistry.
§ Department of Chemistry.
| PROTEOMICS, Interdisciplinary Center for Biotechnology Research
(ICBR).
(3) (a) Kent, S. B. H. Annu. ReV. Biochem. 1988, 57, 957–989. (b) De L.
Milton, R. C.; Milton, S. C. F.; Adams, P. A. J. Am. Chem. Soc. 1990, 112,
6039–6046. (c) Hyde, C.; Johnson, T.; Owen, D.; Quibell, M.; Sheppard, R. C.
Int. J. Peptide Protein Res. 1994, 43, 431–440. (d) Tam, J. P.; Lu, Y. A. J. Am.
Chem. Soc. 1995, 117, 12058–12063.
(4) (a) Chan, W. C.; White, P. D. In Basic Principles: Fmoc Solid Phase
Peptide Synthesis; A Practical Approach; Chan, W. C., White, P. D., Eds.; Oxford
University Press: New York, 2000; pp 32-36. (b) Mergler, M.; Dick, F.; Sax,
B.; Sta¨helin, C.; Vorherr, T. J. Peptide Sci. 2003, 9, 518–526. (c) Palasek, S. A.;
Cox, Z. J.; Collins, J. M. J. Peptide Sci. 2007, 13, 143–148.
(1) (a) Quibell, M.; Johnson, T. In Difficult Peptides: Fmoc Solid Phase
Peptide Synthesis; A Practical Approach; Chan, W. C., White, P. D., Eds.; Oxford
University Press: New York, 2000; pp 115-117. (b) Lloyd-Williams, P.;
Albericio, F.; Giralt, E. Introduction: Chemical Approaches to the Synthesis of
Peptides and Proteins; CRC Press: Boca Raton, FL, 1997; pp 1-17. (c) Cameron,
L. R.; Holder, J. L.; Meldal, M.; Sheppard, R. C. J. Chem. Soc., Perkin Trans.
1 1988, 14, 2895–2901.
(2) (a) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149–2154. (b)
Merrifield, B. Science 1986, 232, 341–347.
2028 J. Org. Chem. 2009, 74, 2028–2032
10.1021/jo8026214 CCC: $40.75 2009 American Chemical Society
Published on Web 02/05/2009

Solid-Phase Assembly of “Difficult” Peptide Sequences
TABLE 1. Preparation of N-Fmoc-(r-Aminoacyl)benzotriazoles
(2) from the corresponding Fmoc-Protected r-Amino Acids (1)
racemized and/or adulterated with deletion sequences or form
aspartimides and related side products.4a,b
literature
yielda
Some of the problems associated with these difficult se-
quences in Fmoc-based SPPS have been alleviated by the use
of (i) bases such as DBU and piperazine (less nucleophilic than
the conventional piperidine) to suppress racemization and to
reduce aspartimide formation;4b,c (ii) chemical ligation tech-
niques, for example the “O-acyl isopeptide method” that can
significantly reduce isomerization of the peptide backbone;5a,b
and (iii) microwave acceleration of the deprotection and
coupling steps,4c,6 which decreases racemization as the growing
peptide has less time available for R-carbon epimerization.
The formation of aspartimides and related side products
remain a problem in SPPS. Backbone protection using the 2,4-
dimethoxybenzyl (Dmb), 2-hydroxy-4-methoxybenzyl (Hmb),
2,4,6-trimethoxybenzyl (Tmb), or 2-nitrobenzyl (Nbzl) groups7a-c
can help but requires additional steps.
entry
compound
(%)
mp (°C)
mp (°C)
ref
1
2
3
4
5
6
7
8
9
Fmoc-L-Val-Bt (2a)
84 151.9-152.6 148.3-149.8 7d
Fmoc-L-Thr(tBu)-Bt (2b)
Fmoc-L-Ser(tBu)-Bt (2c)
Fmoc-L-Tyr(tBu)-Bt (2d)
Fmoc-L-Ile-Bt (2e)
Fmoc-L-Lys(Boc)-Bt (2f)
Fmoc-Gly-Bt (2g)
80
78
84
64.6-66.8 62.2-65.0 7d
63.8-65.4 91.7-92.4b
99.0-100.5 138.4-139.3b
78 165.4-167.2 168.8-170.0 7d
78 138.2-141.7 138.4-140.6 7d
76 161.8-163.3 161.5-161.8 7d
Fmoc-L-Asp(OtBu)-Bt (2h) 81 102.1-104.3 102.0-104.0 7d
Fmoc-L-Phe-Bt (2i) 78 157.0-158.3 159.1-160.2 7d
87
75.3-78.6 121.3-123.2b
10 Fmoc-L-Leu-Bt (2j)
11 Fmoc-L-Met-Bt (2k)
12 Fmoc-L-Ala-Bt (2l)
91 129.1-131.8 122.7-123.3 7d
88 160.5-161.8 160.0-160.3 7d
a Isolated. b See Supporting Information for characterization of the
polymorphs 2c,d,j
Recently, N-Fmoc-(R-aminoacyl)benzotriazoles of proteino-
genic amino acids have been utilized in the synthesis of tri- to
heptapeptides in crude yields of 65-77% on the Rink amide
MBHA solid support.6 We now describe the microwave-assisted
syntheses of six short “difficult” R-peptide sequences in attempts
to examine the extent of racemization, incomplete aminoacy-
lation/deprotection reactions, and the formation of aspartimides
when N-Fmoc-(R-aminoacyl)benzotriazoles are used as activat-
ing reagents for SPPS.
ꢀ-Hydroxy-R-amino acids, such as serine, 3-hydroxyproline,
threonine, and certain analogues (for example, ꢀ-hydroxyphe-
nylalanine and ꢀ-hydroxytyrosine) are widely distributed as
components of biologically active natural products.8a-d The
syntheses of peptides containing ꢀ-hydroxy-R-amino acids can
be challenging (i) due to the risk of racemization of such residues
during both the stepwise and convergent approaches to Fmoc
SPPS5b,9 and (ii) because of aggregation, which may commence
as early as the addition of the fifth amino acid residue in certain
ꢀ-hydroxy-R-amino acid containing sequences.5b,10a,b It is also
known hydrophobic and branched chain amino acids (BCAAs),
such as valine, isoleucine, and leucine, promote aggregation
during peptide synthesis and purification, particularly when a
large percentage of such hydrophobic residues is present.
In the literature the effect of the amino acid hydrophobicity
is evident in the DIPCDI-HOBt (1,3-diisopropylcarbodiimide-
hydroxybenzotriazole) stepwise SPPS of H-Val-Val-Ser-Val-
Val-NH2 (3)5a,b where the undesired N-protected peptide amide
Fmoc-Val-Val-Ser-Val-Val-NH2 was produced as the major
compound (1.1-fold higher than the desired peptide 3 as
evidenced by HPLC5a). In our presently reported work, using
N-Fmoc-(R-aminoacyl)benzotriazoles, peptide 3 was obtained
as the major product (Table 2, Figure 1; Figure 9S, Supporting
Information) with no evidence of the undesired Fmoc-Val-Val-
Ser-Val-Val-NH2 peptide or any racemized product. A possible
explanation for the absence of microaggregates in the benzot-
riazole-assisted synthesis of 3 when compared to the DIPCDI-
HOBt route5a,b could be the impact of microwave irradiation
on the environment of the growing peptide. In our present work,
the alteration of the microenvironment by microwave irradiation
may hinder the formation of insoluble microaggregates and
facilitate the removal of the Fmoc groups from the resin bound
peptide 3.4c Additionally, our microwave-assisted stepwise
protocol reduced the total coupling time for the synthesis of 3
from 105a to 2.5 h (open vessel; 1.5 h when closed vessel
Results and Discussion
N-Fmoc-(R-aminoacyl)benzotriazoles 2a-l (76-91%) were
prepared as previously described7d by treatment of purchased
Fmoc-L-protected amino acids 1a-l with 4 equiv of benzot-
riazole and 1 equiv of SOCl2 in THF at room temperature for
2 h (Scheme 1, Table 1).
SCHEME 1. Preparation of
N-Fmoc-(r-Aminoacyl)benzotriazoles
A standard SPPS approach was employed in the syntheses
of the “difficult” peptides (3-10), in which the appropriate
N-Fmoc-(R-aminoacyl)benzotriazole (2) was coupled in turn to
the growing peptide (Scheme 2). Subsequent cleavage7e from
the Rink amide MBHA resin and purification of the crude
peptide provided the desired product.
(5) (a) Sohma, Y.; Sasaki, M.; Hayashi, Y.; Kimura, T.; Kiso, Y. Chem.
Commun. 2004, 1, 124–125. (b) Sohma, Y.; Taniguchi, A.; Skwarczynski, M.;
Yoshiya, T.; Fukao, F.; Kimura, T.; Hayashi, Y.; Kiso, Y. Tetrahedron Lett.
2006, 47, 3013–3017.
(6) Katritzky, A. R.; Khashab, N. M.; Yoshioka, M.; Haase, D. N.; Wilson,
K. R.; Johnson, J. V.; Chung, A.; Haskell-Luevano, C. Chem. Biol. Drug Des.
2007, 70, 465–468.
(7) (a) Packman, L. C. Tetrahedron Lett. 1995, 36, 7523–7526. (b) Quibell,
M.; Packman, L. C.; Johnson, T. J. Am. Chem. Soc. 1995, 117, 11656–11668.
(c) Miranda, L. P.; Meutermans, W. D. F.; Smythe, M. L.; Alewood, P. F. J.
Org. Chem. 2000, 65, 5460–5468. (d) Katritzky, A. R.; Singh, A.; Haase, D. N.;
Yoshioka, M. ArkiVoc 2009, 47–56. (e) Technical Bulletin, Applied. Biosystems.
(8) (a) Crich, D.; Banerjee, A. J. Org. Chem. 2006, 71, 7106–7109. (b)
Cremonesi, G.; Dalla, Croce, P.; Fontana, F.; Forni, A.; La Rosa, C. Tetrahedron:
Asymmetry 2007, 18, 1667–1675. (c) Davis, F. A.; Srirajan, V.; Fanelli, D. L.;
Portonovo, P. J. Org. Chem. 2000, 65, 7663–7666. (d) Kimura, T.; Vassilev,
V. P.; Shen, G.-J.; Wong, C.-H. J. Am. Chem. Soc. 1997, 119, 11734–11742.
(9) Di Fenza, A.; Tancredi, M.; Galoppini, C.; Rovero, P. Tetrahedron Lett.
1998, 39, 8529–8532.
(10) (a) Bedford, J.; Hyde, C.; Johnson, T.; Jun, W.; Owen, D.; Quibell, M.;
Sheppard, R. C. Int. J. Pept. Prot. Res. 1992, 40, 300–307. (b) Kates, S. A.;
Sole, N. A.; Beyermann, M.; Barany, G.; Albericio, F. Peptide Res. 1996, 9,
106.
(11) (a) Pa´tek, M. Branched-Chain Amino Acids. In Amino Acid Biosynthesis-
Pathways, Regulation and Metabolic Engineering; Wendisch, V. F., Ed.;
Microbiology Monographs; Springer-Verlag: Berlin, Heidelberg, New York,
2007; Vol. 5, pp 129-162. (b) Cornish, V. W.; Kaplan, M. I.; Veenstra, D. L.;
Kollman, P. A.; Schultz, P. G. Biochemistry 1994, 33, 12022–12031.
J. Org. Chem. Vol. 74, No. 5, 2009 2029

Katritzky et al.
SCHEME 2. SPPS Approach Using the Rink Amide MBHA Resin and N-Fmoc-(r-Aminoacyl)benzotriazoles
TABLE 2. Analytical Data of Peptides 3-10
crude
after preparative HPLC
sequence N-C terminus
retention time, tR (min) purity (%) yieldb (%) purity (%)
yield (%) [M + H]+ foundd
H-Val-Val-Ser-Val-Val-NH2 (3)
7.57
9.79
10.82
8.70
8.31
9.10
60
60
70
50
30
37
80
28
63
34
40
35
68
64
86
42
>99
ndc
>99
89
>99
>99
>99
89
7
ndc
24
16
24
29
37
22
501.3385
600.4058
500.3643
618.3864
693.3916
735.4419
555.2948
857.5277
H-Val-Val-Val-Ser-Val-Val-NH2 (4)
H-Val-Ile-Val-Ile-Gly-OHa (5)
H-Thr-Val-Thr-Val-Thr-Val-NH2 (6)
H-Val-Lys-Asp-Gly-Tyr-Ile-NH2 (7)
H-Val-Lys-Asp-Val-Tyr-Ile-NH2 (8)
H-Tyr-Gly-Gly-Phe-Leu-NH2 (9)
11.54
13.82
H-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala-NH2 (10)
a On purification the peptide amide hydrolyzed to the corresponding acid. b Yields quoted are calculated from the amount of product obtained
d
multiplied by % purity from the HPLC. c Not determined. For calculated [M + H]+ values see Supporting Information.
Peptide amides, such as 3 and 4, contain valine, a naturally
occurring branched chain amino acid (BCAA). BCAA com-
pounds occur frequently in the cores of proteins. Such BCAAs
are important in determining the three-dimensional structure of
globular proteins and play a pivotal role in the interactions of
membrane proteins with phospholipid bilayers.11a The absence
of any peaks for the Fmoc protected peptides in both the HPLC
and HRMS spectra of 3 and 4 suggests that interference by
aggregation was negligible in these two examples of our present
work.
We next synthesized peptide 5 (Table 2; Figures 14S and
15S, Supporting Information) possessing 80% BCAAs and with
a hydrophobicity similar to that of 3, in 24% isolated yield,
with no evidence of interference by aggregation (Figure 15S,
Tables 4S and 5S, Supporting Information; during purification
of the peptide the amide was hydrolyzed to the corresponding
acid). Previously, the Boc-based SPPS strategy along with the
DCC/HOBt active ester coupling method was applied in the
synthesis of 5, while our present work utilized the milder Fmoc-
based strategy along with N-(Fmoc-protected-R-aminoacyl)ben-
zotriazoles. A comparison of the yield of 5 via the literature
Boc-based SPPS with our yield by the Fmoc-based benzotria-
zole-assisted strategy could not be calculated from the informa-
tion provided by the previous authors.12
FIGURE 1. HPLC profiles of (a) crude and (b) pure peptide 3 obtained
after SPPS using 20% piperidine-DMF for Fmoc cleavage (open vessel
condition).
conditions are applied, Figure 11S, Supporting Information) and
improved the overall yield from 1.4%5a to 7%. Although using
the O-acyl isopeptide chemical ligation technique to synthesize
3 may provide a peak overall yield of 28% (extrapolated from
the yield of the O-acyl isopeptide of 3), this requires a coupling
time of 40 h.5b
A synthesis analogous to that of 3 gave hydrophobic peptide
4 as the major product (20 mg of 60% purity, comparing to a
yield of pure 4 of 34%), which is further evidence of the utility
of N-Fmoc-(R-aminoacyl)benzotriazoles in the synthesis of
difficult peptides containing ꢀ-hydroxy-R-amino acid residues.
The HPLC and HRMS spectra of crude 4 showed no peaks for
the analogous Fmoc-protected peptide amide or for any race-
mized product (Figures 12S and 13S, Supporting Information).
A literature search for 4 gave no details of the synthesis or yield.
Hexapeptide 6 contains only ꢀ-branched side chains and is
less hydrophobic than 3 and 4. We synthesized compound 6
(Table 2; Figures 16S and 17S, Table 6S, Supporting Informa-
tion) by six successive couplings with (i) Fmoc-Val-Bt, (ii)
Fmoc-Thr(tBu)-Bt, (iii) Fmoc-Val-Bt, (iv) Fmoc-Thr(tBu)-Bt,
(12) Ajikumar, P. K.; Devaky, K. S. J. Pept. Sci. 2001, 7, 641–649.
2030 J. Org. Chem. Vol. 74, No. 5, 2009

Solid-Phase Assembly of “Difficult” Peptide Sequences
FIGURE 2. HPLC profiles of (a) crude and (b) pure peptide 9 obtained after SPPS using 20% piperidine-DMF for Fmoc cleavage.
(v) Fmoc-Val-Bt, and (vi) Fmoc-Thr(tBu)-Bt. The linear
geometry of 6 can be attributed to destabilizing steric effects
and the restricted rotational freedom of peptides containing only
ꢀ-branched amino acids.11b Peptides, such as 6 could be useful
building blocks in tests for the stabilizing or destabilizing effect
of BCAAs in peptide and protein R-helix formation.11b,13 The
nearest literature comparison appears to be the synthesis by
Jolliffe et al.13 of the TBS-protected linear peptide amide (Val-
Thr)3 as an intermediate in the formation of cyclo (Val-Thr)3;
again no direct comparison of yields is possible because of lack
of data in the literature reference.13
In the SPPS of peptides containing asparagine or aspartic acid,
aspartimides are frequently formed.4a,b,14 The proportion of
aspartimide side products depends on the base used for removal
of the Fmoc group,4b the nature of the preceding amino acid
residue,4b,14 and to a lesser extent, the protecting group on the
aspartyl residue.4b,14 We chose to examine hexapeptide 7;4b,15
structure 7 is the peptide fragment 1-6 of toxin II from the
scorpion Androctonus australis Hector16 and contains the Asp-
Gly fragment. Syntheses of compound 7 (Table 2; Figures 18S
and 19S, Supporting Information) and analogue 84b (Table 2;
Figures 20S and 21S, Supporting Information) containing Asp-
Val serve to test the tendency for aspartimide formation using
N-Fmoc-(R-aminoacyl)benzotriazoles. Our synthesis of peptide
amide 7 (m/z 735.4419, tR 8.31 min; Figures 18S and 19S,
Supporting Information) did produce the corresponding as-
partimide (m/z 675.3808, tR 8.11 min; Figures 18S and 19S,
Supporting Information) as a byproduct. However, our synthesis
of peptide amide 8 (Table 2; Figures 20S and 21S, Supporting
Information) proceeded without any of the analogous aspartim-
ide byproduct, the formation of which was evidently suppressed
by replacement of the glycine residue with valine. For both 7
and 8 we used 5% piperazine-DMF (and not 20% piperidine-
DMF) for the removal of the Fmoc group, and this eliminated
ring opening of the aspartimide to the corresponding piperidide
in the case of 7.
Leu-enkephalin (9)17 and Amyloid-ꢀ segment (34-42)
(10).18 We also prepared 9 and 10, two biologically important
“difficult” peptides, by the above strategy. Leu-enkephalin (9)
(Table 2, Figure 2) is a natural peptide neurotransmitter and a
powerful painkiller. For 9 no direct yield comparison with the
literature17 can be made, as no literature yield is provided.
Amyloid-ꢀ protein is the major plaque component in Alzhe-
imer’s disease (AD) and a commercially available product. The
preparation of segment (34-42) (10) (Table 2, Table 10S,
Figure 26S, Supporting Information) was previously described
by Halverson19 and co-workers, who used Kaiser oxime resin
with the alanine residue already attached; they then coupled
N-Boc-protected amino acids eight times to assemble 10, stating
that the solubilization was “extremely difficult” and recovery
subsequent to HPLC analysis was “extremely sensitive” but
claiming a 40% yield of material that was “quite susceptible”
to oxidation.
Conclusions
As summarized in Table 2, our benzotriazole-assisted solid-
phase assembly affords difficult peptides in crude yields of
34-86%. These benzotriazole-assisted syntheses, in tandem with
microwave acceleration, have the following advantages over the
previously used carbodiimide-based coupling methods: (i) our
coupling reactions require no base, (ii) our conditions are
comparatively mild, (iii) our reactions are more rapid, and (iv)
importantly, our products are chirally homogeneous.
1-Hydroxybenzotriazole hydrate (HOBt) is a widely used
coupling additive in peptide synthesis that suppresses racem-
ization when used in combination with carbodiimides such as
DCC. Other 1-hydroxybenzotriazole derivatives, for example,
1-hydroxy-7-azabenzotriazole (HOAt), 6-chloro-1-hydroxyben-
zotriazole (6-Cl-HOBt), phosphonium and aminium salts of
hydroxybenzotriazoles, are also used as additives. Recently, the
availability of HOBt has decreased because of the propensity
for explosion during transportation.20 Thus, this shows the
efficacy of N-Fmoc-(R-aminoacyl)benzotriazoles in peptide
synthesis.
Although 7 was used as a test peptide by four sets of
authors,4b,14-16 no comparison of our yield with the literature
can be made because each literature case reported only product
purity and no yield was provided.4b,14-16 Again, for 8, only
the product purity was provided in the literature,4b making a
yield comparison impossible.
There is a present and growing need for efficient synthetic
methods for the assembly of proteins and peptides needed to
study diverse systems in the body and/or develop a cure for
neurodegenerative diseases such as AD. We have demonstrated
(13) Skropeta, D.; Jolliffe, K. A.; Turner, P. J. Org. Chem. 2004, 69, 8804–
8809.
(17) Thieriet, N.; Guibe´, F.; Albericio, F. Org. Lett. 2000, 2, 1815–1817.
(18) Tickler, A. K.; Clippingdale, A. B.; Wade, J. D. Protein Peptide Lett.
2004, 11, 377–384.
(19) Halverson, K.; Fraser, P. E.; Kirschner, D. A.; Lansbury, P. T.
Biochemistry 1990, 29, 2639–2644.
(14) Wade, J. D.; Mathieu, M. N.; Macris, M.; Tregear, G. W. Lett. Pept.
Sci. 2000, 7, 107–112.
ˇ
(15) Zahariev, S.; Guarnaccia, C.; Pongor, C. I.; Quaroni, L.; Cemazˇar, M.;
Pongor, S. Tetrahedron Lett. 2006, 47, 4121–4124.
(16) Albericio, F.; Nicola´s, E.; Josa, J.; Grandas, A.; Pedroso, E.; Giralt, E.;
Granier, C.; van Rietschoten, J. Tetrahedron 1987, 43, 5961–5971.
(20) Wehrstedt, K. D.; Wandrey, P. A.; Heitkamp, D. J. Hazard. Mater. 2005,
A126, 1–7.
J. Org. Chem. Vol. 74, No. 5, 2009 2031

Katritzky et al.
resulting peptidyl resin was cleaved with cleavage cocktail B7e (88%
TFA/5% phenol/5% water/2% TIPS), K7e (82.5% TFA/5% phenol/
5% water/5% thioanisole/5% EDT), or L7e (88% TFA/5% DTT/
5% water/2% TIPS) for 1.5-2.0 h. Following cleavage, the peptide
was precipitated with cold diethyl ether, and the ether-peptide
mixture was incubated for 24 h at 4 °C and lyophilized to afford
the crude peptides 3-10.
Peptide Analysis. Analyses of the peptides 3-10 (Table 2) were
carried out on 1100 series HPLC equipped with a Phenomenex
Synergi 4u Hydro-RP 80A (2 mm × 150 mm, 4 µm) column plus
a C18 guard column (2 mm × 4 mm). Mass analysis was performed
using a LCQ ion trap mass spectrometer in electrospray ionization
(ESI) mode.
that N-Fmoc-(R-aminoacyl)benzotriazoles are useful for the
solid-phase assembly of “difficult” peptides and represent viable
alternatives as SPPS reagents.
Experimental Section
Solid-Phase Protocol for the Preparation of Peptides 3-10.
Standard stepwise solid-phase synthesis was performed manually
in a 25-mL Discover SPS reaction vessel. Peptides 3-10 were each
prepared on Rink amide MBHA resin. After swelling of the resin
(0.1 mM) in DCM (4 mL, 0.5 h) and treatment with 20%
piperidine-DMF (ca. 3 mL) for 20 min, the solvent was removed,
the free-base amide resin was washed with DMF (5 mL × 3) and
DCM (5 mL × 3), and dried, and a DMF-DCM (5:1 ca. 3 mL)
solution of the appropriate N-Fmoc-(R-aminoacyl)benzotriazole (0.5
mM) was added. The coupling was induced using microwave
irradiation (70-75 °C, 70-80 W, 10-30 min), and the completion
of the coupling reaction was assessed by a negative ninhydrin
(Kaiser) test. Successive N-Fmoc-(R-aminoacyl)benzotriazoles were
similarly coupled to the growing peptide. Deprotection of the
N-Fmoc-(R-aminoacyl)benzotriazole was achieved at each stage
with 20% piperidine-DMF or 5% piperazine-DMF. Finally, the
Acknowledgment. We thank Dr. C. Dennis Hall for his
useful suggestions.
Supporting Information Available: General procedures and
copies of HPLC and mass spectral data. This material is
available free of charge via the Internet at http://pubs.acs.org.
JO8026214
2032 J. Org. Chem. Vol. 74, No. 5, 2009