Solid-phase synthesis of tyrosine peptide aldehydes. Analogues of (S)- MAPI

Article abstract of DOI:10.1021/jo981546v

We report an efficient solid-phase synthesis of C-terminal tyrosine peptide aldehydes based on the HIV protease inhibitors (S)-MAPI and GE 20372 A. Our strategy consisted of anchoring the side chain of Dde-Tyrosinol (5) onto the brominated Wang linker derivative ((4-bromomethyl)-phenoxy-allyl acetate) (6) to give after ester hydrolysis the N^a-(Dde)-O-(4- methylphenoxyacetic acid)-L-Tyrosinol template (8). This was attached to aminomethyl resin and elongated using standard Fmoc protocols. Importantly there was no evidence of esterification side reactions. The unsymmetrically substituted urea linkage of the (S)-MAPI family was incorporated using the N^a-(4-nitrophenyloxycarbonyl)amino acid tert-butyl esters following which the protected tetrapeptide alcohol immobilized on the solid support was oxidized to its corresponding aldehyde using sulfur trioxide-pyridine. The efficiency and reliability of the oxidation step was dramatically improved by the incorporation of a small PEG-spacer between the linker and the solid support. The tetrapeptides 12a and 12b were cleaved by acidolysis, purified by RP HPLC, and isolated in high yield and purity, demonstrating the success of the whole synthetic process.

Full text of DOI:10.1021/jo981546v

794
J . Org. Chem. 1999, 64, 794-799
Solid -P h a se Syn th esis of Tyr osin e P ep tid e Ald eh yd es. An a logu es
of (S)-MAP I
Patrick Page and Mark Bradley*
Department of Chemistry, University of Southampton, Southampton SO17 1BJ , UK
Iain Walters and Simon Teague
Department of Medicinal Chemistry, Astra Charnwood, Bakewell Road, Loughborough,
Leicestershire, LE11 ORH, UK
Received August 3, 1998
We report an efficient solid-phase synthesis of C-terminal tyrosine peptide aldehydes based on the
HIV protease inhibitors (S)-MAPI and GE 20372 A. Our strategy consisted of anchoring the side
chain of Dde-Tyrosinol (5) onto the brominated Wang linker derivative ((4-bromomethyl)-phenoxy-
allyl acetate) (6) to give after ester hydrolysis the NR-(Dde)-O-(4-methylphenoxyacetic acid)-L-
Tyrosinol template (8). This was attached to aminomethyl resin and elongated using standard
Fmoc protocols. Importantly there was no evidence of esterification side reactions. The unsym-
metrically substituted urea linkage of the (S)-MAPI family was incorporated using the NR-(4-
nitrophenyloxycarbonyl)amino acid tert-butyl esters following which the protected tetrapeptide
alcohol immobilized on the solid support was oxidized to its corresponding aldehyde using sulfur
trioxide-pyridine. The efficiency and reliability of the oxidation step was dramatically improved
by the incorporation of a small PEG-spacer between the linker and the solid support. The
tetrapeptides 12a and 12b were cleaved by acidolysis, purified by RP HPLC, and isolated in high
yield and purity, demonstrating the success of the whole synthetic process.
In tr od u ction
to be widely exploited medicinally. For these reasons,
recent literature provides many examples of the biological
activity of peptide aldehydes toward a variety of biological
targets. Thus, peptide aldehyde Ac-Leu-Ala-Ala-(N,N′-
dimethylglutaminal) was found to be a reversible, slow-
binding inhibitor for hepatitis-A virus (HAV) 3C protein-
ase8 with a Ki of 42 nM. Whereas R-MAPI (or (S)-MAPI,
Ki ) 1.3 µM) (1) was described by Stefanelli et al.9 as a
potent inhibitor of the human immunodeficiency virus
(HIV) despite it being an aspartic acid protease.10 This
protease is essential for the processing of the viral
polyproteins and plays a crucial role in the HIV life cycle.
Related to MAPI is GE 20372 A which was recently
isolated from Streptomyces sp. ATCC 55925 (Ki ) 18 µM),
and which also demonstrates activity against HIV pro-
tease.11 This family of tetrapeptides has two common
features: a terminal aldehyde and a urea link between
two amino acid residues.12
Serine1,2 and cysteine3,4 proteases are potently inhib-
ited by peptide aldehydes1 and peptide trifluoromethyl
ketones.5 Inhibition arises from the formation of hemi-
acetal or hemithioacetal tetrahedral adducts within the
active sites of the proteases which mimic the proposed
structure of the transition state/intermediate.3 The C-
terminal peptide aldehydes are thus an important class
of transition-state analogues and have been the focus of
considerable attention since their initial discovery in
natural products.6 Importantly, many serine and cysteine
proteases have been implicated in a number of disease
states,7 and thus, their inhibition has been and continues
(1) (a) Woo, J . T.; Sigeizumi, S.; Yamagushi, K. Bioorg. Med. Chem.
Lett. 1995, 5, 1501-1504. (b) Schacht, A. L.; Wiley: M. R.; Chirgadze,
N.; Clawson, D.; Craft, T. J .; Coffman, W. J . Bioorg. Med. Chem. Lett.
1995, 5, 2529-2534.
(2) (a) Bullock, T. L.; Breddam, K.; Remington, S. J . J . Mol. Biol.
1996, 255, 714-725. (b) Schultz, R. M.; Varma-Nelson, P.; Ortiz, R.;
Kozlowsky, K. A.; Orawsky, A. T.; Pagast, P.; Frankfater, A. J . Biol.
Chem. 1989, 264, 1497-1507. (c) Kennedy, W. P.; Schultz, R. M.
Biochemistry 1979, 18, 349-356. (d) Thompson, R. C. Biochemistry
1973, 12, 47-51.
(3) (a) Mackenzie, N. E.; Grant, S. K.; Scott, A. I.; Malthouse, J . P.
G. Biochemistry 1986, 25, 2293-2298. (b) Schroder, E.; Phillips, C.;
Garmen, E.; Harlos, K.; Crawford, C. FEBS Lett. 1993, 315, 38-42.
(4) (a) Bendal, M. R.; Cartwright, I. L.; Clark, P. I.; Lowe, G. Eur.
J . Biochem. 1977, 79, 201-209. (b) Frankfater, A.; Kuppy, T. Bio-
chemistry 1981, 20, 5517-5524.
(5) Ogilvie, W.; Bailey, M.; Poupart, M.-A.; Abraham, A.; Bhavsar,
A.; Bonneau, P.; Bordeleau, J .; Bousquet, Y.; Chabot, C.; Duceppe, J .;
Fazal, G.; Goulet, S.; Grand-Maitre, C.; Guse, I.; Halmos, T.; Lavallee,
P.; Leach, M.; Malenfant, E.; O’Meara, J .; Plante, R.; Plouffe, C.;
Poirier, M.; Soucy, F.; Yoakim, C.; Deziel, R. J . Med. Chem. 1997, 40,
4113-4135.
(7) Aoyagi, T.; Umezawa, H. In Proteases and Biological Control;
Reich, E., Rifkin, D. B., Shaw, E., Eds.; Cold Spring Harbor Press:
Cold Spring Harbor, NY, 1975; pp 429-454.
(8) Malcolm, B. A.; Lowe, C.; Shechosky, S.; Mckay, R. T.; Yang, C.
C.; Shah, V. J .; Simon, R. J .; Vederas, J . C.; Santi, D. V. Biochemistry
1995, 34, 8172-8179.
(9) Stella, S.; Saddler, G.; Sarubbi, E.; Colomb, L.; Stefanelli, S.;
Denaro, M.; Selva, E. J . Antibiot. 1991, 44, 1019-1022.
(10) (a) Sarubbi, E.; Seneci, P. F.; Angelastro, M. R.; Peet, N. P.;
Denaro, M.; Islam, K. FEBS Lett. 1993, 319, 253-256. (b) Zhang, X.;
Rodrigues, J .; Evans, L.; Hinkle, B.; Ballantyne, L.; Pena, M. J . Org.
Chem. 1997, 62, 6420-6423.
(11) Stefanelli, S.; Cavaletti, L.; Sarubbi, E.; Ragg, E.; Colombo, L.;
Selva, E. J . Antibiot. 1995, 48, 332-334.
(6) (a) Aoyagi, T.; Takeuchi, T.; Matsuzaki, A.; Kawamura, M.;
Kondo, M.; Hamada, M.; Maeda, K.; Umezawa, H. J . Antibiot. 1969,
22, 283. (b) Westerik, J . O.; Wolfenden, R. J . Biol. Chem. 1972, 247,
8195-8197.
(12) (a) Kaneto, R.; Chiba, Dobashi,.; Kojima, I.; Sakai, K.; Shiba-
moto, N.; Nisshida, H.; Okamoto, R.; Akagawa, H.; Mitsuno, S. J .
Antibiot. 1993, 46, 1622-1624. (b) Watanbe, T.; Murao, S. Agric. Biol.
Chem. 1979, 43, 243-250.
10.1021/jo981546v CCC: $18.00 © 1999 American Chemical Society
Published on Web 01/16/1999

Solid-Phase Synthesis of Tyr Peptide Aldehydes
J . Org. Chem., Vol. 64, No. 3, 1999 795
chain and followed the reported solid-phase oxidation
methods of Kurth.20a Thus the overall strategy was based
on the side-chain anchoring of a tyrosinol derivative onto
the widely utilized Wang linker, providing an ideal
template for attachment to aminomethyl resins, followed
by subsequent Fmoc peptide chemistry, urea formation,
alcohol oxidation, and finally compound cleavage from
the solid support.
Our initial synthetic approach envisioned a Mitsunobu
reaction between the Wang linker derivative (4-hy-
droxymethyl)phenoxyallyl acetate and Fmoc-Tyrosinol.21
The allyl ester functionality could then be removed
orthogonally to the Fmoc group prior to solid phase
immobilization. Unfortunately, all attempts to carry out
this Mitsunobu reaction, using a variety of coupling
reagents such as DEAD/PPh3, TMAD/PBu3, and solvents
(THF, DCM, and NMM) were unsuccessful, and no
coupling products were isolated. Although formation of
triphenyl phosphine oxide was observed after a few
minutes when DEAD/PPh3 were used as activating
agents, purification of the crude reaction mixture on silica
gel yielded pure starting materials. O-Alkylation reac-
tions using the brominated Wang linker derivative22 were
also unsuccessful with the Fmoc-Tyrosinol derivative due
to extensive Fmoc removal in the presence of bases such
as cesium carbonate, sodium hydrogen carbonate, sodium
hydride, or 1 equiv of potassium hexamethyl disilazane.
F igu r e 1. Human immunodeficiency virus (HIV) aspartic acid
protease inhibitors GE 20372 and MAPI.
Resu lts a n d Discu ssion
Ongoing work within our group on urea containing
peptides as inhibitors of serine proteases attracted us to
the (S)-MAPI and GE 20372 A (Figure 1) family of
peptide aldehydes. Since the C-terminal residue was
phenylalanine, we decided to use the corresponding
hydroxylated residue tyrosine as this would allow side-
chain anchoring to a solid support via an appropriate
linker. The unsymmetrical substituted urea linkage of
the (S)-MAPI family would be synthesized using NR-(4-
nitrophenyloxycarbonyl)amino acid esters,13 although
Pena et al.10b has recently published a solution synthesis
of urea dipeptides of this class of compounds using
carbonyldiimidazole. Several methods for the solid-phase
synthesis of peptide aldehydes have been reported.14-19
Bray et al.14 described an oxazolidine linker based on
threonine or serine, and Murphy et al.15 developed a
linker based on semicarbazone amino acid derivatives,
although both of these methods required the use of
protected amino acid aldehydes. Other linkers and
techniques have also been devised, for example linkers
have been developed on the basis of the Weinreb amide,16
although they give low cleavage yields and the final
products were contaminated with LiAlH4 byproducts,
applications were found in the synthesis of both peptide17
and small molecule aldehydes.18 Recently, J ouin et al.19
reported the preparation of peptide aldehydes by copper
salt-mediated neutral hydrolysis of the corresponding
C-terminal thiazolidinyl peptides.
We therefore switched to the synthetic strategy shown
in Scheme 1 which utilized as a key compound Dde-
Tyrosinol (5). This was prepared in 3 steps from
commercially available Fmoc-Tyrosinol(tBu) (3) by
Fmoc removal followed by N-reprotection with 2-acetyl
dimedone23 and tertiary butyl ether cleavage with
trifluoroacetic acid in an overall yield of 86%. The NR-
(Dde)-O-(4-methylphenoxyacetic acid)-L-Tyrosinol scaffold
(8) was prepared in high yield (91%) by treatment of (5)
with the brominated Wang linker22 derivative (6) in the
presence of cesium carbonate in refluxing acetonitrile
followed by ester hydrolysis, either by specific allyl ester
removal with Pd(0) and dimedone24 or by classical
saponification which was obviously more appropriate for
the production of the free carboxylic acid scaffold on a
large scale.
The free carboxylic acid (8) was coupled to amino-
methyl-(poly(ethylene glycol)-polystyrene-resin) (amino-
methyl-PEG-PS-resin) (9) (which was synthesized in
multigram quantities from aminomethyl-PS-resin in
three steps as shown in Scheme 2), using diisopropyl-
carbodiimide (DIC)/hydroxybenzotriazole (HOBt) in dichlo-
romethane/THF and gave an initial loading of 0.65
mmol/g (98% expected) as determined by quantitative
Dde analysis.23b The Dde group was removed with a 10%
solution of hydrazine in DMF to give the free amine.23a
We decided to develop an efficient method based on a
resin-supported oxidation of a peptide alcohol. The
method, if successful, would be very versatile and ap-
plicable to any amino alcohol with a resin attachable side
(13) Marsh, I. R.; Bradley, M.; Teague, S. J . Org. Chem. 1997, 62,
6199-6203.
(14) Ede, N. J .; Bray, A. M. Tetrahedron Lett. 1997, 38, 7119-7122.
(15) Murphy, A. M.; Dagnino, R., J r.; Vallar, P. L.; Trippe, A. J .;
Sherman, S. L.; Lumpkin, R. H.; Tamura, S. V.; Webb, T. R. J . Am.
Chem. Soc. 1992, 114, 3156-3157.
(20) (a) Chen, C.; Ahlberg-Randall, L. A.; Miller, R. B.; J ones, A.
D.; Kurth, M. J . J . Am. Chem. Soc. 1994, 116, 2661-2662. (b) Rotella,
D. P. J . Am. Chem. Soc. 1996, 118, 12246-12247.
(21) (a) Richter, L. S.; Gadek, T. R. Tetrahedron Lett. 1994, 35,
4705-4706. (b) Rano, T. A.; Chapman, K. T. Tetrahedron Lett. 1995,
36, 3789-3792.
(16) Nahm, S.; Weinreb, S. Tetrahedron Lett. 1981, 22, 3815-3818.
(17) Fehrentz, J .-A.; Paris, M.; Heitz, A.; Velek, J .; Liu, C.-F.;
Winternitz, F.; Martinez, J . Tetrahedron Lett. 1995, 36, 7871-7874.
(18) Dinh, T. Q.; Armstrong, R. W. Tetrahedron Lett. 1996, 37,
1161-1164.
(22) Ngu, K.; Patel, D. V. Tetrahedron Lett. 1997, 38, 973-976.
(23) (a) Bloomberg, G. B.; Askin, D.; Gargaro, A. R.; Tanner, M. J .
A. Tetrahedron Lett. 1993, 34, 4709. (b) Bycroft, B. W.; Chan, W. C.;
Chhabra, S. R.; Hone, N. D. J . Chem. Soc. Chem. Commun. 1993, 778-
779.
(24) Kunz, H.; Unverzagt, C. Angew. Chem., Int. Ed. Engl. 1984,
23, 436.
(19) Galeotti, N.; Giraud, M.; J ouin, P. Lett. Pept. Sci. 1997, 4, 437-
440.

796 J . Org. Chem., Vol. 64, No. 3, 1999
Page et al.
Sch em e 1a
a
Conditions: (i) 40% Et2NH, DCM, 98%; (ii) Dde-OH, triethylamine, DMF, 90%; (iii) 50% trifluoracetic acid, DCM, 98%; (iv) 6, Cs2CO3,
acetonitrile reflux, 89%; (v) NaOH, THF/H2O (9:1), 97% or Pd(PPh3)4, dimedone, THF/DCM (1:1), 94%; (vi) 9, DIC, HOBt, DCM/THF; (vii)
10% NH2NH2/DMF; (viii) Fmoc peptide synthesis, Fmoc deprotection, 20% piperidine/DMF; sequential coupling with (a) Fmoc-Val-OH,
DIC, HOBt, DCM/DMF; (b) Fmoc-Arg(Boc)2-OH, DIC, HOBt, DCM/DMF; (ix) 10a or 10b, DIPEA, DMF; (x) SO3-Pyr, triethylamine,
anhydrous DMSO (×2); (xi) 95% trifluoracetic acid, 5% phenol or water.
Sch em e 2. P r ep a r a tion of
poor quantification of the amines on the amino alcohol
rather than to failed couplings. We attribute this to the
poor cleavage of the ninhydrin imine formed when
ninhydrin reacts with the amino alcohol.
Am in om eth yl-P EG-p olystyr en e Resin (9)a
Fmoc chemistry continued with the addition of Fmoc-
L-Arg(Boc)2-OH and Fmoc removal. The urea functional-
ity was incorporated efficiently using 1.1 equiv of NR-(4-
nitrophenyloxycarbonyl)-O-(tert-butyl)-L-tyrosine-tert-
butyl ester (10a ) in DMF/diisopropyl ethylamine13 and
was monitored by both the quantitative ninhydrin test
and by removal of small quantities of resin (ca. 5 mg),
trifluoroacetic acid cleavage, and HPLC (shown in Figure
2a) and ES (electrospray) MS analysis.26
Conversion of the totally protected tetrapeptide alcohol
immobilized on solid support (11a ) to its corresponding
aldehyde was achieved using a sulfur trioxide-pyridine
mediated oxidation.20 Of the various oxidation methods
examined, which included Dess-Martin periodinane,
activated manganese dioxide, and acetic anhydride/
DMSO, 10 equiv of sulfur trioxide-pyridine in anhydrous
dimethyl sulfoxide20 in the presence of triethylamine
turned out to be the most reliable. An important observa-
tion was that the oxidation failed when polystyrene resin
was utilized but that the use of the aminomethyl-poly-
(ethylene glycol)-polystyrene resin (9) dramatically im-
a
Conditions: (i) DCC, DCM; (ii) aminomethyl polystyrene resin,
DCM; (iii) Boc-NH(CH2)2NH2, DIC, HOBt, DCM; (iv) 50% trifluo-
racetic acid/DCM.
At this stage a classical ninhydrin test25 gave a loading
of 25% of the expected value. Continuation of peptide
synthesis (Fmoc-Val-OH) then gave Fmoc and ninhydrin
values of the full, and expected, value, indicating that
the poor ninhydrin value obtained previously related to
(26) Typical HPLC-ES MS procedure: A 5-8 mg sample of resin
was preswollen in dichloromethane and subjected to treatment with
trifluoracetic acid/water (95:5, 1 mL) for 1 h. After filtration and
evaporation of the solvents, the product was analyzed by HPLC and
ES MS.
(25) Sarin, V. K.; Kent, S. B. H.; Tam, J . P.; Merrifield, R. B. Anal.
Biochem. 1981, 20, 147-157.

Solid-Phase Synthesis of Tyr Peptide Aldehydes
J . Org. Chem., Vol. 64, No. 3, 1999 797
Exp er im en ta l Section
Gen er a l P r oced u r es. Reactions in solution were moni-
tored by thin-layer chromatography on precoated aluminum-
backed plates (Merck silica gel 60 F254). The chromatograms
were visualized by UV light and staining with phosphomo-
lybdic acid (PMA), bromocresol green, permanganate, or
ninhydrin. All reagents and solvents were obtained from
commercial suppliers and used without further purification.
Specific sources of reagents were as follows: Fmoc-Tyrosinol-
(tBu), Advanced ChemTech; 3,6,9-trioxaundecanedioic acid,
Fluka; H-Tyr(tBu)-OtBu‚HCl and H-Phe-OtBu‚HCl, NovaBio-
chem; TentaGel S AM (0.26 mmol/g), Rapp Polymer; Merrifield
resin (1.34 mmol/g) based on 1% cross-linked divinylbenzene
styrene copolymer (100-200 mesh) was purchased from Nova-
Biochem and was treated with potassium phthalimide and
hydrazine to give the corresponding aminomethyl polystyrene
resin.27 Analytical RP-HPLC was performed on a Phenomenex
Prodigy ODS3 5 µm C18 column (3 × 150 mm) with a linear
gradient of acetonitrile/0.043% trifluoracetic acid (0-100% over
20 min) in water/0.1% trifluoracetic acid at a flow rate of 0.5
mL/min and monitoring at 220 nm.
F igu r e 2. Crude RP-HPLC (C-18) of (a) tetrapeptide alcohol
cleavage; (b) tetrapeptide aldehyde (12a ), and (c) purified
tetrapeptide aldehyde (12a ).
proved the efficiency of the reaction. No tetrapeptide
aldehyde (12a ) was obtained when the corresponding
primary alcohol (11a ), anchored to aminomethyl poly-
styrene resin was subjected to five treatments with 10
equiv of sulfur trioxide-pyridine/triethylamine in anhy-
drous dimethyl sulfoxide. The incorporation of the amino-
PEG-spacer between the linker, and the solid support
obviously reduces the steric hindrance in the environ-
ment of the primary alcohol functionality and improves
the solvation of the solid-phase support. The use of
TentaGel S aminomethyl resin was also successful and
gave (12a ) after one treatment with 10 equiv of sulfur
trioxide-pyridine in anhydrous dimethyl sulfoxide in the
presence of triethylamine and a few activated 4 Å
molecular sieves. However, the substitution of our resin
(9) (0.97 mmol/g) is much higher than TentaGel and thus
was favored.
Nr-(Dd e)-L-Tyr osin ol(tBu ) (4). To a solution of 40%
diethylamine in DCM (50 mL) was added Fmoc-Tyrosinol(tBu)
(2.57 g, 5.77 mmol), and the mixture was stirred for 3 h. The
reaction mixture was concentrated under reduced pressure,
the residue was dissolved in diethyl ether (6 mL), and water
(20 mL) was added. This organic/aqueous solution was con-
centrated under reduced pressure to remove the ether phase.
The resulting aqueous layer was diluted with water (50 mL),
and the precipitate was collected by filtration. The filtrate was
lyophilized, giving a white solid which was redissolved in DCM
(50 mL), dried over MgSO4, and filtered, and the solvent was
removed in vacuo to give H-L-Tyrosinol(tBu) as a white foam
1
(1.26 g, yield 98%). H NMR (300 MHz, CDCl3) δ: 1.28 (9H,
s), 2.47 and 2.68 (1H + 1H, AB part of ABX system, J AX ) 6
Hz, J BX ) 7 Hz J AB ) 13 Hz), 3.05 (1H, br m), 3.28 (3H, br m),
3.32 and 3.56 (1H + 1H, AB part of ABX system, J AX ) 4 Hz,
J BX ) 8 Hz J AB ) 11 Hz), 6.85 (2H, d, J ) 8 Hz), 7.02 (2H, d,
J ) 8 Hz). 13C NMR (75 MHz, CDCl3) δ: 28.97 (CH3), 39.42
(CH2), 54.42 (CH), 65.43 (CH2), 78.48 (C), 124.46 (CH), 129.74
(CH), 133.19 (C), 154.03 (C). A solution of 2-acetyldimedone
(1.16 g, 6.34 mmol) in DMF (10 mL) and triethylamine (1 mL,
6.92 mmol) were added to a solution of H-Tyrosinol(tBu) (1.26
g, 5.65 mmol) in DMF (5 mL), and the mixture was stirred
overnight. The reaction mixture was poured into EtOAc (150
mL), the solution was washed with 2 M KHSO4 (2 × 50 mL)
and brine (50 mL), and the organic layer was dried over
MgSO4, filtered. The solvent was removed in vacuo to give an
orange solid. The residue was purified by column chromatog-
raphy on silica gel eluting with hexane/EtOAc (2:1) to give the
title compound as a yellow-orange solid (2.01 g, yield 90%).
1H NMR (300 MHz, CDCl3) δ: 0.94 (6H, s), 1.24 (9H, s), 2.15
(4H, s), 2.2 (3H, s), 2.7 and 2.9 (1H + 1H, AB part of ABX
system m), 3.7 (2H, m), 3.97 (1H, m), 4.3 (1H, m), 6.83 (2H, d,
J ) 7 Hz), 6.99 (2H, d, J ) 7 Hz), 13.49 (1H, m br). 13C NMR
(75 MHz, CDCl3) δ: 17.88 (CH3), 28.27 (CH3), 28.88 (CH3), 30.0
(C), 37.86 (CH2), 52.68 (CH2), 57.82 (CH), 63.91 (CH2), 78.65
(C), 107.88 (C), 124.56 (CH), 129.84 (CH), 131.95 (C), 154.27
(C), 173.66 (C), 197.98 (C). ES MS m/ e: 388.4 (M + H)+.
HRMS for C23H33O4N: calcd 387.2410, found 387.2427.
Nr-(Dd e)-L-Tyr osin ol (5). To a solution of compound (4)
(2.0 g, 5.17 mmol) in DCM (20 mL) was added trifluoroacetic
acid (20 mL), and the reaction was stirred at room temperature
for 40 min. The solution was diluted with DCM (60 mL) and
washed with brine (2 × 25 mL). The organic layer was dried
over MgSO4 and filtered, and the solvent was removed in vacuo
to give a yellow oil. This oil was purified by column chroma-
tography on silica gel eluting with DCM/MeOH (95:5) to give
Compound 12a was cleaved from the solid support
using trifluoracetic acid/phenol (95:5) for 2 h. After
evaporation of the solvents, precipitation with tert-butyl
methyl ether and centrifugation, the crude product (12a )
was dissolved in water/acetonitrile and lyophilized before
purification by reverse-phase HPLC. The overall yield of
the whole solid-phase synthetic process (9 steps, includ-
ing anchoring and cleavage steps) was 55-70%, and the
crude tetrapeptide (12a ) was 68% pure as determined
by HPLC analysis (and ES MS) (Figure 2b). There was
1
no evidence of diastereoisomers by either H or 13C NMR
analysis. Finally, the tetrapeptide (12b) was isolated
using a similar procedure to that of 12a , except the urea
functionality was incorporated by means of NR-(4-nitro-
phenyloxycarbonyl)-L-phenylalanine-tert-butyl ester (10b).
In conclusion, we have devised a simple and very
efficient solid-phase synthesis of C-terminal tyrosine
peptide aldehydes. NR-(Dde)-O-(4-methylphenoxyacetic
acid)-L-Tyrosinol (8) provides an ideal scaffold that can
be easily attached to solid supports and elongated using
Fmoc protocols. Furthermore, we demonstrated that the
use of immobilized amino-PEG-polystyrene resin (9)
improves dramatically the efficiency of the oxidation
reaction and that this resin is inexpensive and easy to
prepare. Finally, the synthesis reported herein, for a
C-terminal tyrosine analogue of the natural product (S)-
MAPI may provide a novel template leading to thera-
peutic agents in the treatment of AIDS.
1
a colorless oil (1.68 g, yield 98%). H NMR (300 MHz, CDCl3)
δ: 0.94 (6H, s), 2.24 (4H, s), 2.28 (3H, s, CH3), 2.68 and 2.88
(1H + 1H, AB part of ABX system m), 3.7 (3H, m), 3.98 (1H,
(27) Weinshenker, N. M.; Shen, C. M. Tetrahedron Lett. 1972, 32,
3281-3284.

798 J . Org. Chem., Vol. 64, No. 3, 1999
Page et al.
m), 6.73 (2H, d, J ) 7 Hz), 6.97 (2H, d, J ) 7 Hz), 13.47 (1H,
d, J ) 8 Hz). 13C NMR (75 MHz, CDCl3) δ: 18.26 (CH3), 28.24
(CH3), 30.2 (C), 37.54 (CH2), 52.52 (CH2), 57.81 (CH), 63.66
(CH2), 107.92 (C), 115.87 (CH), 127.67 (C), 130.47 (CH), 155.98
(C), 174.09 (C), 198.58 (C). ES MS m/ e: 332.2 (M + H)+.
HRMS for C19H25O4N: calcd 331.1785, found 331.1784.
Nr-(Dd e)-O-(4-m eth ylp h en oxy-a llyl a ceta te)-L-Tyr osi-
n ol (7). Dde-Tyrosinol (5) (1.68 g, 5.07 mmol), Cs2CO3 (2.34
g, 7.19 mmol) and the bromo linker derivative (6) (2.2 g, 7.7
mmol) in acetonitrile (100 mL) were refluxed for 3 h. The
resulting solution was concentrated under reduced pressure,
the residue redissolved in EtOAc (200 mL), washed with brine
(2 × 60 mL), and filtered, and the solvent was removed in
vacuo to give a yellow oil which was purified by column
chromatography on silica gel eluting with DCM/MeOH (94:6)
30 mL) and DCM (3 × 30 mL). A ninhydrin test showed
complete coupling. To this resin was added a solution of DIC
(1.72 mL, 11.04 mmol) in DCM (15 mL), and the reaction
mixture was shaken for 30 min before mono-Boc-protected
ethylenediamine (1.8 g, 11.04 mmol) was added and the
solution was shaken overnight. The resin was filtered and
washed with DMF (3 × 30 mL) and DCM (3 × 30 mL). A BCG-
test showed complete coupling. The resin was shaken in 50%
trifluoracetic acid/DCM for 30 min, filtered, and washed with
DCM (3 × 30 mL), 10% DIPEA/DMF (3 × 30 mL), DMF (3 ×
30 mL), MeOH (3 × 30 mL), and ether (3 × 30 mL), and the
resin was dried in vacuo overnight. A quantitative Fmoc test
gave a loading of 0.97 mmol/g (93% expected).
Tetr a p ep tid e (12a ). Diisopropylcarbodiimide (61 µL, 0.388
mmol) was added to a solution of compound (8) (192 mg, 0.388
mmol) and HOBt (53 mg, 0.388 mmol) in 4 mL THF/DCM (1:
1). The preactivated carboxylic acid scaffold was added to
aminomethyl-PEG-polystyrene resin (9) (200 mg, 0.194 mmol,
preswollen for 30 min in DCM), and the mixture was shaken
for 4 h. The resin was filtered, washed with DMF (3 × 4 mL),
DCM (3 × 4 mL), MeOH (3 × 4 mL), and ether (3 × 4 mL),
and dried in vacuo. Resin substitution was determined by
quantitative Dde analysis and found to be 0.65 mmol/g (98%
expected). This resin was preswollen in DMF (3 mL) for 15
min and filtered, and a solution of 10% hydrazine monohydrate
in DMF (4 mL) was added. The reaction mixture was shaken
for 1 h. The resin was filtered, washed with DMF (3 × 4 mL),
DCM (3 × 4 mL), and subjected to Fmoc-peptide synthesis
using the following conditions: (i) Fmoc deprotection, 20%
piperidine in DMF (4 mL) for 30 min, followed by washing with
DMF (3 × 4 mL) and DCM (3 × 4 mL); (ii) coupling conditions,
(a) Fmoc-Val-OH (132 mg, 0.388 mmol), HOBt (53 mg, 0.388
mmol), and DIC (61 µL, 0.388 mmol) in DCM/DMF (4 mL:10
drops), 4 h; (b) Fmoc-Arg(Boc)2-OH (231 mg, 0.388 mmol),
HOBt (53 mg, 0.388 mmol), and DIC (61 µL, 0.388 mmol) in
DCM/DMF (4 mL:10 drops), 6 h; following all couplings the
resin was filtered and washed with DMF (3 × 4 mL) and DCM
(4 × 4 mL). All couplings were analyzed using the ninhydrin
test.
NR-(4-Nitrophenyloxycarbonyl)-O-(tert-butyl)-L-tyrosine-tert-
butyl ester13 (10a ) (100 mg, 0.218 mmol) and DIPEA (40 µL,
0.218 mmol) were dissolved in DMF (3 mL) and added to the
peptide resin, and the reaction was shaken for 1 h. The resin
was filtered, washed with DMF (3 × 4 mL), DCM (3 × 4 mL),
MeOH (4 × 4 mL), and ether (3 × 4 mL), and dried overnight
in vacuo. A ninhydrin test showed complete coupling. A
solution of sulfur trioxide-pyridine (309 mg, 1.94 mmol) and
triethylamine (270 µL, 194 mmol) in 4 mL of commercially
available anhydrous DMSO (99.8% from Aldrich) was added
to the resin (preswollen for 30 min in anhydrous DMSO), and
the reaction mixture was shaken overnight. The resin was
filtered, washed with anhydrous DMSO (3 × 4 mL), and
followed by a second treatment with sulfur trioxide-pyridine
and triethylamine in anhydrous DMSO. The resin was filtered
and washed with DMSO (6 × 4 mL), DCM (3 × 4 mL), and
ether (3 × 4 mL). The efficiency of the oxidation step was
assessed by acid cleavage of a few milligrams of resin and LC/
MS analysis. The product was cleaved from the solid support
by treatment with trifluoracetic acid/phenol (95:5, 3 mL) or
trifluoracetic acid/water (95:5, 3 mL) for 2 h. The resin was
filtered and washed with trifluoracetic acid (2 × 2 mL), and
the filtrate and washes were combined. The solution was
concentrated in vacuo (ca. 0.5-1 mL), and 15 mL of tert-butyl
methyl ether was added. After 15 min centrifugation (5000
rpm/ 25 °C), a white-yellow solid was isolated which was
redissolved in water with a few drops of acetonitrile and
lyophilized (67 mg, 55% yield from starting resin loading). The
product was 68% pure as determined by analytical HPLC
analysis. Purification was achieved using semipreparative RP
HPLC on a Phenomenex Prodigy ODS 5 µm 250 × 10 mm
column, eluting at 2.5 mL/min. The following gradient was
used: t ) 0 min, 100% A; t ) 30 min, 25% A, 75% B; t ) 35
min, 100% B (A ) water, 0.1% trifluoracetic acid, B )
acetonitrile, 0.043% trifluoracetic acid). Under these conditions
1
to give a colorless oil (2.42 g, yield 89%). H NMR (300 MHz,
CDCl3) δ: 0.99 (6H, s), 2.27 (4H, s), 2.29 (3H, s), 2.73 and 2.94
(1H + 1H, AB part of ABX system, J AX ) 6 Hz, J BX ) 7 Hz,
J AB ) 13.5 Hz), 3.72 (2H, m), 3.90 (1H, m), 3.99 (1H, br m),
4.65 (2H, s), 4.69 (2H, d, J ) 5 Hz), 4.92 (2H, s), 5.30 (2H,
ddd, J ) 1.5 Hz, J ) 11.5 Hz, J ) 17 Hz), 5.92 (1H, ddd, J )
7 Hz, J ) 11.5 Hz, J ) 17 Hz), 6.84 (2H, d, J ) 8 Hz), 6.91
(2H, d, J ) 8 Hz), 7.04 (2H, d, J ) 8 Hz), 7.33 (2H, d, J ) 8
Hz), 13.55 (1H, d, J ) 9 Hz). 13C NMR (75 MHz, CDCl3) δ:
18.0 (CH3), 28.34 (CH3), 30.15 (C), 37.59 (CH2), 52.81 (CH2),
57.73 (CH), 63.98 (CH2), 65.49 (CH2), 66.03 (CH2), 69.73 (CH2),
107.95 (C), 114.91 (CH), 115.17 (CH), 119.32 (CH2), 129.14 (C),
129.4 (CH), 130.25 (C), 130.42 (CH), 131.52 (CH), 157.74 (C),
157.88 (C), 168.73 (C), 173.69 (C), 198.07 (C). ES MS m/ e:
536.4 (M + H)+. HRMS for C31H37O7N: calcd 536.2648, found
536.2617.
Nr-(Dd e)-O-(4-m eth ylp h en oxya cetic a cid )-L-Tyr osin ol
(8). Meth od A. Allyl ester (7) (440 mg, 0.822 mmol) was
dissolved in THF/DCM (1:1, 100 mL), and the solution was
stirred and degassed for 1 h with a gentle flow of nitrogen
through the solution. Dimedone (682 mg, 4.92 mmol) and Pd-
(PPh3)4 (144 mg, 0.124 mmol) were added, and the reaction
was stirred under nitrogen for 3 h at room temperature. The
solution was concentrated in vacuo, and the residue was
redissolved in DCM (200 mL). The organic layer was washed
with 2 M KHSO4 (2 × 50 mL), brine (50 mL), and finally dried
over MgSO4 and concentrated in vacuo to give a yellow-orange
residue. The residue was purified by column chromatography
on silica gel eluting with DCM/MeOH (96:4) to give a white
foam (398 mg, 97%).
Meth od B. Compound (7) (3.56 g, 6.65 mmol) was dissolved
in THF (90 mL), and a solution of sodium hydroxide (293 mg,
7.32 mmol) in distilled water (10 mL) was added. The reaction
mixture was stirred for 50 min at room temperature. The
solution was concentrated under reduced pressure to ca. 10
mL, poured into water (20 mL), and acidified with 2 M KHSO4.
The aqueous phase was extracted with EtOAc (300 mL, 6 ×
50 mL) and DCM (150 mL, 3 × 50 mL), the extracts were
combined, washed (brine, 60 mL), dried (MgSO4), and filtered,
and the solvent was removed in vacuo to give a white foam
1
(3.1 g, yield 94%). H NMR (300 MHz, CDCl3) δ: 1.0 (6H, s),
2.2 (3H, s), 2.28 (4H, s), 2.68 and 2.87 (1H + 1H, AB part of
ABX system m), 3.69 (2H, m), 3.93 (1H, br m), 4.55 (2H, s),
4.85 (2H, s), 6.77 (2H, d, J ) 8 Hz), 6.82 (2H, d, J ) 7 Hz),
6.95 (2H, d, J ) 7 Hz), 7.24 (2H, d, J ) 8 Hz), 7.75 (1H, br s),
13.45 (1H, d, J ) 8 Hz). 13C NMR (75 MHz, CDCl3) δ: 18.34
(CH3), 28.29 (CH3), 30.24 (C), 37.46 (CH2), 52.41 (CH2), 57.8
(CH), 63.67 (CH2), 65.35 (CH2), 69.7 (CH2), 107.89 (C), 114.86
(CH), 115.29 (CH), 129.06 (C), 129.37 (CH), 130.07 (C), 130.44
(CH), 157.74 (C), 157.80 (C), 171.68 (C), 174.23 (C), 198.6 (C).
ES MS m/ e: 496.5 (M + H)+, 534.5 (M + K)+. HRMS for
C
28H33O7N: calcd 496.2335, found 496.2369.
P r ep a r a tion of Am in om eth yl-P EG-p olystyr en e Resin
(9). A solution of 3,6,9-trioxaundecanedioic acid (5 g, 22.5
mmol) and DCC (4.64 g, 22.5 mmol) was stirred in DCM (100
mL) for 6 h at room temperature. The reaction mixture was
filtered, concentrated in vacuo (ca. 30 mL), and added to
aminomethyl polystyrene resin (1 g, loading 1.38 mmol/g,
preswollen for 30 min in DCM), and the mixture was shaken
overnight. The resin was filtered and washed with DMF (3 ×

Solid-Phase Synthesis of Tyr Peptide Aldehydes
J . Org. Chem., Vol. 64, No. 3, 1999 799
the product eluted at 21.8 min. Yield: 39 mg. 1H NMR (360
MHz, DMSO-d6) δ: 0.85 (6H, m), 1.52 (3H, m), 1.71 (1H, m),
2.00 (1H, m), 2.67 (1H, dd, J ) 7 Hz, J ) 13 Hz), 2.82 (2H, m),
2.97 (1H, dd, J ) 5 Hz J ) 13 Hz), 3.21 (2H, td, 2xJ ) 6 Hz),
4.07 (1H, m), 4.11 (1H, dd, J ) 7 Hz, J ) 9 Hz), 4.26-4.40
(2H, m), 6.30 (1H, d, J ) 8 Hz), 6.59 (1H, d, J ) 9 Hz), 6.74
(2H, d, J ) 8 Hz), 6.75 (2H, d, J ) 8 Hz), 7.04 (2H, d, J ) 8
Hz), 7.09 (2H, d, J ) 8 Hz), 7.52 (1H, t, J ) 6 Hz), 7.66 (1H,
d, J ) 8 Hz), 7.98 (1H, d, J ) 9 Hz), 9.28 (1H, br s), 12.63 (1H,
br s). 13C NMR (75 MHz, DMSO-d6) δ: 17.75 (CH3), 19.15
(CH3), 24.87 (CH2), 30.33 (CH2), 30.67 (CH), 35.78 (CH2), 36.68
(CH2), 40.35 (CH2), 50.08 (CH), 52.25 (CH), 54.04 (CH), 58.37
(CH), 114.94 (CH), 114.95 (CH), 127.1 (C), 128.35 (C), 129.99
(CH), 130.11 (CH), 155.59 (C), 155.95 (C), 156.56 and 156.98
(Cx2), 170.32 and 171.94 (2xC), 173.74 (C), 200.23 (CH); ES
MS m/ e: 628.7 (aldehyde form) (M + H)+, 646.7 (M + H2O +
H)+.
Tetr a p ep tid e (12b). The title compound was synthesized
following the same procedure described for tetrapeptide 12a .
ES MS m/ e: 612.8 (aldehyde form) (M + H)+, 630.4 (M + H2O
+ H)+, 634.9 (M + Na)+. HRMS for C30H43O8N7: calcd
630.3251, found 630.3199.
Ack n ow led gm en t. The authors thank Astra Charn-
wood for financial support and the Royal Society for a
University Research Fellowship (M.B.).
J O981546V