Synthesis of the palmitoylated and prenylated C-terminal lipopeptides of the human R- and N-Ras proteins

Bioorganic & Medicinal Chemistry 7 (1999) 749±762

Synthesis of the Palmitoylated and Prenylated C-terminal

Lipopeptides of the Human R- and N-Ras Proteins

T. Schmittberger and H. Waldmann*

Universitat Karlsruhe, Institut fur Organische Chemie, Richard-Willstatter-Allee 2, D-76128 Karlsruhe, Germany

È È

È

Received 7 August 1998; accepted 28 September 1998

AbstractÐFor the study of biological phenomena in¯uenced by the R- and N-Ras proteins, characteristic peptides which embody

the correct lipid modi®cations of their parent proteins (palmitoyl thioesters, geranylgeranyl thioethers, and farnesyl thioethers), as

well as analogues thereof, may serve as ecient tools. For the construction of such acid- and base labile peptide conjugates the allyl

ester was developed as C-terminal protecting group. Allyl esters are cleaved selectively and in high yields from lipidated peptides by

Pd(0)-mediated allyl transfer to accepting N- or C-nucleophiles like morpholine and N,N⁰-dimethylbarbituric acid. This protecting

group technique formed the key step in the synthesis of the characteristic S-palmitoylated and S-isoprenylated C-terminus of human

R-Ras and human N-Ras proteins, as well as several analogues thereof. Deprotections are so mild that no undesired side reactions

Introduction

cal mechanism by which cells can actively regulate their

own and/or each other's death, thereby preventing

damaged cells from harming the organism.

Lipid modi®ed proteins are critically involved in the

transduction of stimuli from the extracellular space

across the plasma membrane into the cell, and ulti-

mately to the cell nucleus.^1,2The biological signi®cance

of lipidated proteins is highlighted by the crucial roles of

the so-called Ras proteins in maintaining the regular life

cycle of cells. H-, N-, and K-Ras proteins are plasma-

membrane bound lipoproteins which are found in

organisms as diverse as mammals, ¯ies, worms and

yeast. They serve as central molecular switches,^3,4and

translate the signals given by growth factors via a well-

balanced series of non-covalent protein/protein interac-

tions into a cascade of highly speci®c protein phos-

phorylations, resulting in the activation of transcription

factors (Scheme 1). Thus, Ras regulates cell growth and

proliferation. If this regulation is disturbed or inter-

rupted, uncontrolled proliferation may occur which

may result in transformation of the cell. Thus, a point

mutation in the ras oncogenes coding for the Ras pro-

teins is found in ca. 30% of all human cancers, a ®gure

which rises up to 80% for some of the major malig-

nancies like pancreas cancer.⁵Furthermore, the R-Ras

protein may have a key regulatory role in a signal

transduction pathway involved in the regulation of

programmed cell death⁶(apoptosis), i.e. the physiologi-

All Ras proteins are post transitionally modi®ed.⁷In

this process precursor proteins are S-prenylated, then

the three C-terminal amino acids are proteolized and

the C-terminal amino acid is converted into the methyl

ester. Finally, further cysteine residues may be S-palmi-

toylated. Thus, H-, N- and K-Ras are S-farnesylated,

and H-, and N-Ras carry additional palmitic acid

thioesters (Scheme 2). The R-Ras protein is S-geranyl-

geranylated and S-palmitoylated (Scheme 2). H-, N-

and K-Ras must be lipid-modi®ed and plasma mem-

brane associated to perform both their normal and

oncogenic biological functions. In the non lipidated

forms the Ras proteins are cytosolic and are not able to

transduce signals,^3,8and it is believed that the interac-

tion between Ras and its downstream eector Raf

(Scheme 1) is mediated at least in part via the farnesyl

group present in Ras.^9,10The lipidated C-terminus of R-

Ras was shown to associate with the apoptosis-suppres-

sing proto-oncogene product Bcl-2,¹¹suggesting a role

for R-Ras in the regulation of programmed cell death

(see above).

The ®nding that lipidation can be crucial to guarantee

correct cellular signaling may have far reaching con-

sequences for biological and pharmaceutical research. It

may provide important clues for a better understanding

of biological signal transduction, as well as improve the

eciency of drugs which in¯uence signal transduction

processes.^5,12

Key words: Ras proteins; signal transduction; lipidated peptides;

protecting groups; allyl esters.

*Corresponding author. Tel: +49 721 608 2091; fax: +49 721 608

4825; e-mail: waldmann@ochhades.chemie.uni.karlsruhe.de

PII: S0968-0896(98)00251-X

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T. Schmittberger, H. Waldmann / Bioorg. Med. Chem. 7 (1999) 749±762

For the study of biological phenomena which are in¯u-

enced by lipid-modi®ed proteins the combination of

techniques of cell biology, organic synthesis and bio-

physics opens up new and alternative opportunities.^13±19

For such studies characteristic lipidated peptides

embodying the correct lipid groups and amino acid

sequences of their parent lipid-modi®ed proteins are

useful reagents.^15±18Such compounds can for instance

shed light on cellular mechanisms by which these cova-

lently modi®ed polypeptides may be targeted to their

subcellular destination, i.e. the plasma membrane.^17,18,20

Unfortunately, the synthesis of such peptide conjugates

as for instance the characteristic C-terminal lipidated

peptides 1 and 2 of human R-Ras and N-Ras is severely

complicated by their pronounced acid- and base lability

(Scheme 2).^13,21±25Thus, during acid-mediated removal

of the Boc group from S-farnesylated cysteinyl peptides,

an attack of the acid on the double bonds of the farnesyl

residue always occurs,²¹whereas the thioesters present

in S-palmitoylated lipidated peptides hydrolyze sponta-

neously even at pH 6±7 in aqueous solution.^22±24Due to

this pronounced acid and base lability, dierent ortho-

gonally stable blocking functions have to be employed

that can be selectively removed under the mildest

conditions.²⁶

Allyl-based protecting groups have proven to be versa-

tile tools for the synthesis of complex, multifunctional

and sensitive compounds.²⁷In general, they can be

removed by means of a Pd(0)-mediated allyl transfer to

accepting nucleophiles like morpholine 3 and N,N⁰-

dimethylbarbituric acid 4 under very mild conditions

(see Scheme 3 for a description of the general process as

exempli®ed for allyl esters). In particular, the use of allyl

ester type protecting groups²⁸has allowed the synthesis

of complex, sensitive glycopeptides in solution and on

the solid phase.^27,29These advantageous properties of

allyl-type blocking functions have prompted us to

investigate their application in the synthesis of S-acy-

lated and S-prenylated peptides. We now report that

characteristic lipidated peptides which represent lipid

modi®ed substructures of the human R- and N-Ras

proteins as well as analogues thereof can be built up

eciently by employing the allyl ester as protecting

group.³⁰

Results and Discussion

Synthesis of the S-palmitoylated and S-geranyl

geranylated C-terminus of human R-Ras protein

For the construction of an S-palmitoylated and S-

geranylgeranylated R-Ras peptide⁷and analogues to 1,

the cystinyl tripeptide allyl ester 10 was built up as the

N-terminal building block (Scheme 4). To this end, Boc-

glycine 5 was coupled with glycine allyl ester 6. From

the resulting fully protected dipeptide 7 the allyl ester

was removed selectively by Pd(0)-mediated allyl transfer

to morpholine 3. Dipeptide acid 8 was then condensed

with cystine bis(allyl ester) 9 to yield the desired inter-

mediate 10. Reduction of the disul®de 10 with dithio-

threitol and subsequent treatment of the resulting

cysteine peptide with palmitoyl chloride gave S-

palmitoylated tripeptide allyl ester 11. The thioester

Scheme 1. The Ras signal transduction pathway.

Scheme 2. Structures of the characteristic acid and base labile C-

terminal lipopeptides of human N- and R-Ras proteins.

Scheme 3. Removal of allyl protecting groups by Pd(0) mediated allyl

transfer to accepting nucleophiles.

T. Schmittberger, H. Waldmann / Bioorg. Med. Chem. 7 (1999) 749±762

751

is weakly C H acidic, thus basic conditions are pre-

vented during the deprotection step.

Upon treatment of tripeptide allyl ester 11 with

Pd(PPh₃)₄in the presence of C-nucleophile 4 the C-

terminal carboxylic acid was smoothly deprotected

without any harm to the base-sensitive thioester bond to

yield tripeptide carboxylic acid 12 in excellent yield.

Thus N,N⁰-dimethylbarbituric acid promises to be a

generally useful allyl accepting nucleophile for the

removal of allyl-type blocking groups from lipidated

peptides. Elongation of the peptide chain by proline

allyl ester 13 and a further Pd(0)-mediated allyl ester

cleavage in the presence of C-nucleophile 4 delivered the

selectively unmasked S-palmitoylated tetrapeptide car-

boxylic acid 14 in high yield. Finally, the synthesis was

completed by coupling of acid 14 with S-geranylger-

anylated cysteine methyl ester 15³²to give the desired R-

Ras peptide 16.

The allyl ester cleavages performed in the sequence

shown in Scheme 4 proceeded with complete selectivity

and without any undesired side reactions. The condi-

tions of the noble-metal complex mediated allyl transfer

are so mild that neither an attack on the base-sensitive

thioester nor a base-induced b-elimination of the pal-

mitoyl group occurred.

Synthesis of the S-palmitoylated and S-farnesylated

C-terminus of human N-Ras protein

The strategy underlying the synthesis of the R-Ras pep-

tide described above proved also very fruitful for the

synthesis of N-Ras peptides and analogues thereof.

Thus, ®rst S-palmitoylated and C-terminally unmasked

dipeptide 19 was built up (Scheme 5). To this end, Boc-

glycine 5 was coupled with cystine bis(allyl ester) 9 to

yield disul®de 17. Reductive cleavage of the disul®de

and subsequent S-palmitoylation as described above

delivered S-acylated dipeptide allyl ester 18. Encouraged

by the eciency of the allyl ester deprotection under

weakly acidic conditions (see above) we investigated

whether the noble-metal complex mediated selective

removal of this blocking group from sensitive S-palmi-

toylated peptides might also be feasible under weakly

basic conditions. Morpholine 3 is an N-nucleophile with

a relatively low pK_Avalue (8.4). It was advantageously

applied in the removal of allyl-type protecting groups in

glycopeptide chemistry.^29,30Therefore, the use of this

nitrogen base as allyl-trapping reagent in the unmasking

of S-allylated peptide 18 was investigated. Upon treat-

ment of allyl ester 18 with Pd(PPh₃)₄in the presence of

morpholine the allyl ester protecting group was split o

without any undesired side reaction and dipetide build-

ing block 19 was obtained in high yield (Scheme 5). No

b-elimination or attack on the thioester was detected.

Thus morpholine also promises to be a generally useful

allyl-accepting nucleophile for the construction of base

labile S-acylated lipopeptides. The peptide chain was

then elongated by condensation of carboxylic acid 19

with N-terminally unmasked tripeptide allyl ester 29a

(for synthesis of 29a see Scheme 6). Upon treatment of

S-palmitoylated pentapeptide allyl ester 20 obtained

Scheme 4. Synthesis of S-palmitoylated and S-geranylgeranylated C-

terminal pentapeptide 16 of human R-Ras protein.

present in tripeptide 11 is very sensitive to nucleophilic

attack.^22,24Therefore, the choice of the allyl-accepting

nucleophile in the projected Pd(0)-catalyzed removal of

the allyl ester protecting group is crucial to the success-

ful realization of the entire synthetic strategy. The allyl-

trapping reagent must be nucleophilic enough to attack

the intermediary formed p-allylpalladium complex

(Scheme 3). It should not induce a b-elimination of the

entire palmitic acid or attack the activated thioester

itself. For the selective removal of allyl protecting

groups a variety of nucleophiles is available that may

ful®ll these criteria.²⁷To solve the challenging problem

posed by the selective C-terminal deprotection of 11, we

used N,N⁰-dimethylbarbituric acid 4 as nucleophile.

This compound has already proven to be an ecient

allyl-trapping reagent in glycopeptide chemistry.^27,31It

752

T. Schmittberger, H. Waldmann / Bioorg. Med. Chem. 7 (1999) 749±762

thereby with a Pd(0) catalyst in the presence of

C-nucleophile 4 the C-terminal allyl ester protecting

group was cleaved o smoothly to give rise to penta-

peptide carboxylic acid 21 in high yield. Further chain

elongation by proline allyl ester 13 and subsequent allyl

ester cleavage yielded selectively deprotected hexapep-

tide carboxylic acid 23. In order to complete the synth-

esis of N-Ras- peptide 25, the S-palmitoylated and

C-terminally deblocked hexapeptide 23 was condensed

with S-farnesylated cysteine methyl ester 24.^21,33As in

the synthesis of the R-Ras peptide detailed above, all

allyl ester cleavage reactions proceeded without b-elim-

ination of palmitic acid or attack on the thioester bond.

post-translational modi®cation of the Ras proteins. As

described above, N-Ras peptide 25 essentially was built

up from four building blocks: a) an S-palmitoylated

dipeptide b) a central tripeptide c) a proline and d)

S-farnesylated cysteine methyl ester (see Scheme 5). By

varying the structure of either one of these building

blocks, but keeping the sequence of coupling and

deprotection steps unchanged, analogues of Ras peptide

25 should be readily accessible.

To this end, the amino acid sequence of the central tri-

peptide unit was varied. Thus, tripeptide allyl esters

29a±d were built up by condensation of amino acid allyl

esters 27 with Boc-protected glycine dipeptide car-

boxylic acids 26 and subsequent removal of the Boc

group from the intermediary formed tripeptides 28

(Scheme 6). Boc protected, C-terminally unmasked

dipeptides 26, were synthesized by condensation of the

corresponding Boc amino acid with glycine allyl ester 6

and subsequent removal of the allyl group (see the

Experimental).

Synthesis of S-palmitoylated and S-farnesylated

analogues of the C-terminus of N-Ras

Analogues of N-Ras peptide 25 may be useful tools for

exploring the precise role of the N-Ras C-terminus for

instance in membrane binding or in processing and

Coupling of S-palmitoylated dipeptide building block

19 with the N-terminally deprotected tripeptide allyl

esters 29b±d yielded lipopeptides 30a±c in high yields

(Scheme 7). The allyl ester protecting group could be

removed from esters 30a±c employing the Pd(0) medi-

ated deprotection technique described in detail above.

To give pentapeptide carboxylic acids 31a±c in high

yields. In the case of 31a and 31b the peptide chain was

then elongated by proline allyl ester and the C-terminus

of the resulting palmitoylated hexapeptides 32 was once

more deprotected by Pd(0) catalyzed allyl transfer to

N,N⁰-dimethylbarbituric acid. Finally, the carboxylic

acids 33a,b obtained thereby were condensed with

S-farnesylated cysteine methyl ester 24 to yield the

desired analogues 35a and 35b of the N-Ras peptide. In

Scheme 5. Synthesis of S-palmitoylated and S-farnesylated C-terminal

heptapeptide 25 of human N-Ras protein.

Scheme 6. Synthesis of tripeptide allyl esters 29.

T. Schmittberger, H. Waldmann / Bioorg. Med. Chem. 7 (1999) 749±762

753

conditions of the noble-metal complex-mediated trans-

fer of the allyl group to accepting N- or C-nucleophiles

like morpholine and N,N⁰-dimethylbarbituric acid are

so gentle that no undesired side reactions occur. In

addition to the results described above for allyl esters we

have recently obtained evidence that also the Pd(0)-

mediated selective removal of N-allyloxycarbonyl ure-

thane protecting groups from lipidated peptides can be

achieved successfully.²⁴Taken together these ®ndings

demonstrate that allyl-type protecting groups advanta-

geously complement the recently developed enzymatic

techniques for synthesis of S-acylated and S-farnesy-

lated peptides^18,22,23that proceed under extremely mild

conditions. By means of these protecting group strate-

gies²⁶further functionalized lipidated peptides carrying

e.g. ¯uorescent labels or biotin units by which they can

be traced in biological systems may be synthesized e-

ciently.^17,18

Experimental

General. ¹H NMR spectra were recorded on Bruker AC

250 (250 MHz), Bruker AM 400 (400 MHz), and Bruker

DRX 500 (500 MHz) NMR spectrometers. ¹³C NMR

spectra were recorded on Bruker AC 250 (62.8 MHz),

Bruker AM 400 (100.6 MHz), and Bruker DRX 500

(125.6 MHz) NMR spectrometers. Optical rotations

were recorded on Perkin±Elmer 241 digital polarimeter.

Melting points were measured on Heraeus CHN-rapid

melting point apparatus without correction.

N-tert-Butyloxycarbonyl-glycyl-glycine allyl ester (7).

To a solution of Boc-protected glycine 5 (1.75 g,

10 mmol) and glycine allyl ester hydro-p-toluenesulfo-

nate 6 (2.96 g, 10 mmol) in CH₂Cl₂(30 mL) was added

1-hydroxybenzotriazole (2.70 g, 20 mmol) and triethyla-

mine (1.38 mL, 10 mmol). After stirring for 5 min at

room temperature, diisopropylcarbodiimide (1.70 mL,

11 mmol) was added. The mixture was allowed to stir

for 12 h at room temperature, and then was washed with

1 N hydrochloric acid (20 mL), with aqueous (5%)

sodium bicarbonate (20 mL) and ®nally with water

(2Â20 mL). The organic layer was dried over magne-

sium sulfate, ®ltered and the solvent was removed in

vacuo. The resulting crude product was puri®ed by col-

umn chromatography on silica gel (hexane/ethyl acet-

ate, 2/3 v/v) to aord 7 (2.21 g, 81%) as a colorless oil:

R_f=0.55 (hexane/ethyl acetate, 2/3 v/v); ¹H NMR

(CDCl₃, 250 MHz) d 1.48 (s, 9H, CH₃), 3,85 (d, 2H,

J=6.2 Hz, CH₂-NH), 4.12 (d, 2H, J=4.5 Hz, CH₂-

NH), 4.65 (d, 2H, J=5.1 Hz, CH₂-C-O), 5.10±5.23 (m,

3H, HN urethane+H₂CC), 5.82±6.01 (m, 1H,

HCC), 6.72 (m br, 1H, HN); ¹³C NMR (CDCl₃,

62.8 MHz) d 28.3 (CH₃), 41.2 (CH₂-NH), 44.1 (CH₂-

NH), 66.1 (CH₂-C-O), 80.2 (Cq), 119.0 (H₂C), 131.4

(HC), 156.2 (CO urethane), 169.5 (CO), 170.1

(CO).

Scheme 7. Synthesis of lipidated peptides 35 that are analogues to N-

Ras peptide 25.

addition, selectively deprotected pentapeptide 31c was

directly condensed with N-terminally deprotected far-

nesylated dipeptide ester 34²¹to give analogue 35c. All

Pd(0) catalyzed allyl ester cleavage reactions performed

in these reaction sequences proceeded smoothly and

without undesired side reactions. In no case could a b-

elimination or a nucleophilic attack on the activated

thioesters be observed.

Conclusion

The use of the allyl ester as C-terminal protecting group

makes sensitive S-palmitoylated and S-isoprenylated

peptides corresponding to the C-terminus of human R-

and N-Ras protein available in an ecient manner. The

N-tert-Butyloxycarbonyl-glycyl-glycine (8). Dipeptide

allyl ester 7 (1.60 g, 5.9 mmol) was dissolved in THF

(50 mL) at room temperature under argon. Tetra-

kis(triphenylphosphine) palladium (10 mg) and 5 min

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T. Schmittberger, H. Waldmann / Bioorg. Med. Chem. 7 (1999) 749±762

later morpholine (0.56 mL, 6.4 mmol) were added. The

mixture was stirred at room temperature for 1 h. The

solvent was removed in vacuo, and the resulting oil was

dissolved in aqueous (2.5%) sodium bicarbonate

(30 mL). The aqueous layer was extracted with Et₂O

(2Â20 mL). The pH was adjusted to 2 with 1 N hydro-

chloric acid and extracted with ethyl acetate (3Â20 mL).

The combined organic layers were dried over magne-

sium sulfate, ®ltered and the solvent was evaporated in

vacuo. The dipeptide 8 was obtained as a white solid

(930 mg, 68%): R_f=0.80 (ethyl acetate/MeOH, 4/1 v/v);

v/v); [a]²_d²13 (c 1.0, CH₂Cl₂); ¹H NMR (CDCl₃,

400 MHz) d 0.90 (t, 3H, J=7.0 Hz, CH₃Pal), 1.25 (s br,

24H, CH₂Pal), 1.45 (s, 9H, CH₃Boc), 1.65 (m, 2H,

CH₂-Me Pal), 2.55 (t, 2H, J=7.5 Hz, CH₂-CO Pal),

3.35 (dd, 1H, J=14.0 Hz, J=6.0 Hz, HC(H)-S), 3.43

(dd, 1H, J=14.0 Hz, J=4.5 Hz, HC(H)-S), 3.85 (m, 2H,

CH₂-NH), 3.90±3.95 (dd, 1H, J=17 Hz, J=5.5 Hz,

HC(H)-NH), 4.00±4.10 (dd, 1H, J=17 Hz, J=5.5 Hz,

HC(H)-NH), 4.60 (m, 2H, 2H, CH₂-O), 4.75 (m, 1H,

CH-NH), 5.25±5.30 (dd, 1H, J=10.5 Hz, J=1.5 Hz,

H(H)CC), 5.30±5.35 (dd, 1H, J=17.0 Hz, J=1.5 Hz,

H(H)CC), 5.45 (t br, 1H, NH urethane), 5.80±6.00

(m, 1H, HC), 7.00 (s br, 1H, NH), 7.10 (s br, 1H,

NH); ¹³C NMR (CDCl₃, 100.5 MHz) d 14.0 (CH₃Pal),

22.7 (CH₂Pal), 25.5 (CH₂Pal), 28.3 (CH₃Boc), 29.0±

30.0 (12 CH₂Pal), 31.9 (CH₂-S), 42.7 (CH₂-NH), 44.0

(CH₂-NH), 52.7 (CH-NH), 66.5 (CH₂-O), 80.3 (Cq),

119.1 (H₂CC), 131.3 (HCC), 156.0 (CO urethane),

168.8 (CO), 169.5 (CO), 170.0 (CO), 199.7 (CO

thio ester); Anal. calcd for C₃₁H₅₅N₃O₇S (613.80): C,

60.66; H, 9.03; N, 6.85. Found: C, 60.47; H, 8.75; N,

7.02.

mp 125±126 ^ꢀC (lit.³⁴125±127 ^ꢀC); H NMR (MeOD,

1

400 MHz) d 1.40 (s, 9H, CH₃), 3.65 (s, 2H, CH₂-NH),

3,80 (s, 2H, CH₂-NH); ¹³C NMR (MeOD, 100.5 MHz)

d 29.2 (CH₃), 42.9 (CH₂-NH), 44.4 (CH₂-NH), 79.7

(Cq), 157.4 (CO urethane), 171.3 (CO), 171.6

(CO).

Bis-(N-tert-butyloxycarbonyl-glycyl-glycyl)-^L-cystine bis-

(allyl ester) (10). 1-Hydroxybenzotriazole (827 mg,

6.12 mmol) and triethylamine (0.43 mL, 3.06 mmol)

were added to a suspension of 8 (710.0 mg, 3.06 mmol)

and cystine bis(allyl ester) hydro-p-toluenesulfonate 9

(917 mg, 1.53 mmol) in CH₂Cl₂/DMF (10/1, 15 mL).

After stirring for 5 min at room temperature, diisopro-

pylcarbodiimide (0.52 mL. 3.36 mmol) was added. The

mixture was allowed to stir for 12 h at room tempera-

ture, and then was washed with 1 N hydrochloric acid

(10 mL), with aqueous (5%) sodium bicarbonate

(10 mL), and ®nally with water (2Â10 mL). The organic

layer was dried over magnesium sulfate, ®ltered and the

solvent was removed in vacuo. The resulting crude pro-

duct was puri®ed by column chromatography on silica

gel (MeOH/ethyl acetate, 1/10 v/v) to aord 10 (780 mg,

81%) as a colorless viscous oil: R_f=0.35 (MeOH/ethyl

acetate, 1/10 v/v); [a]²_d²52^ꢀ(c 1.20, CH₂Cl₂); ¹H

NMR (CDCl₃, 250 MHz) d 1.47 (s, 18H, CH₃), 2.95±

3.25 (m, 4H, CH₂-S), 3.80±4.15 (m, 8H, 2ÂCH₂-NH),

4.63 (d, 4H, J=5,7 Hz, CH₂-O), 4.80±4.92 (m, 2H, HC-

NH), 5.22±5.40 (m, 4H, H₂CC), 5.65 (m br, 2H, NH

urethane), 5.80±5.97 (m, 2H, HCC), 7.35 (m br, 2H,

NH), 7.52 (m br, 2H, NH); ¹³C NMR (CDCl₃,

62.8 MHz) d 28.1 (CH₃), 41.4 (CH₂-NH), 44.2 (CH₂-

NH), 47.8 (CH₂-S), 51.7 (HC-NH), 66.6 (CH₂-O), 80.9

(Cq), 119.1 (H₂CC), 131.4 (HCC), 156.3 (CO

urethane), 169.8 (CO), 170.2 (CO), 170.7 (CO).

N-tert-Butyloxycarbonyl-glycyl-glycyl-(S-palmitoyl)-^L-

cysteine (12). Tetrakis(triphenylphosphine) palladium

(5 mg) and 1,3-dimethyl-pyrimidone-2,4,5-trione 4 (85.4 mg,

0.55 mmol) were added under argon atmosphere to a

solution of 11 (560 g, 0.91 mmol) in THF (10 mL) at

room temperature. The resulting mixture was stirred at

room temperature for 1 h. The solvent was removed in

vacuo, and the resulting oil was dissolved in CH₂Cl₂

(5 mL) and hexane was added until all the product pre-

cipitated. 12 was isolated as a yellow solid (474 mg,

91%) and used without further puri®cation: R_f=0.15

(ethyl acetate/MeOH, 10/1 v/v); [a]²_d²16^ꢀ(c 1.54,

1

CH₂Cl₂); H NMR (CDCl₃, 250 MHz) d 0.91 (t, 3H,

J=6.9 Hz, CH₃Pal), 1.25 (s br, 24H, 12 CH₂Pal), 1.47

(s, 9H, CH₃Boc), 1.61±1.73 (m, 2H, CH₂-Me Pal), 2.62

(t, 2H, J=7.5 Hz, CH₂-CO Pal), 3.29 (dd, 1H,

J=14.0 Hz, J=6.1 Hz, HC(H)-S), 3.44 (dd, 1H,

J=14.0 Hz, J=4.5 Hz, HC(H)-S), 3.81±3.96 (m, 3H,

CH₂-NH+HC(H)-NH), 4.05±4.20 (m, 1H, HC(H)-

NH), 4.68 (m, 1H, CH-NH), 5.60 (m br, 1H, NH ure-

thane), 7.35 (m br, 2H, 2ÂNH); ¹³C NMR (CDCl₃,

62.8 MHz) d 14.1 (CH₃Pal), 22.2 (CH₂Pal), 25.5 (CH₂

Pal), 28.3 (CH₃Boc), 28.8±30.3 (12 CH₂Pal), 31.9

(CH₂-S), 42.8 (CH₂-NH), 44.0 (CH₂-NH), 52.6 (CH-

NH), 66.5 (CH₂-O), 80.3 (Cq), 157.2 (CO urethane),

169.4 (CO), 170.0 (CO), 173.2 (CO), 200.1 (CO

thio ester).

N-tert-Butyloxycarbonyl-glycyl-glycyl-(S-palmitoyl)-^L-

cysteine allyl ester (11). Triethylamine (0.32 mL,

2.28mmol) and 1,4-dl-dithiothreitol (527mg, 3.42mmol)

were added under argon atmosphere to a solution of 10

(780 mg, 1.04 mmol) in CH₂Cl₂(30 mL). After stirring

for 2 h at room temperature, the solution was washed

with 1 N hydrochloric acid (20 mL), with water

(2Â20 mL), and dried over sodium sulfate. The solution

was ®ltered and cooled to 0 ^ꢀC. Triethylamine (0.32 mL,

2.28 mmol) and palmitoyl chloride (1.38 mL, 4.56 mmol)

were added. The mixture was allowed to stir for 2 h at

room temperature. After ®ltration and evaporation of

CH₂Cl₂in vacuo, a crude viscous oil was obtained,

which was puri®ed by column chromatography on silica

gel (ethyl acetate). Compound 11 was isolated as a white

solid (827 mg, 65%): R_f=0.20 (hexane/ethyl acetate, 1/1

N-tert-Butyloxycarbonyl-glycyl-glycyl-(S-palmitoyl)-^L-

cysteyl-^L-proline (14). 1-Hydroxybenzotriazole (80.3 mg,

0.592 mmol) and triethylamine (41.3 mL, 0.296 mmol)

were added to a suspension of tripeptide 12 (170 mg,

0.296 mmol) and proline allyl ester hydro-p-toluene-

sulfonate 13 (100 mg, 0.296 mmol) in CH₂Cl₂(10 mL).

After stirring for 5 min at room temperature, N-(3-di-

methylaminopropyl)-N⁰-ethylcarbodiimide hydrochloride

(85.2 mg, 0.444 mmol) was added. The mixture was

allowed to stir for 12 h at room temperature. Then it

was washed with 1 N hydrochloric acid (5 mL), with

aqueous (5%) sodium bicarbonate (5 mL), and ®nally

T. Schmittberger, H. Waldmann / Bioorg. Med. Chem. 7 (1999) 749±762

755

with water (2Â5 mL). The organic layer was dried over

magnesium sulfate, ®ltered and the solvent was evapo-

rated in vacuo. The resulting oil was dissolved in anhy-

drous THF (5 mL) at room temperature under argon

atmosphere, tetrakis(triphenyl-phosphine) palladium

(2 mg) and 5 min later 1,3-dimethyl-pyrimidone-2,4,5-

trione 4 (6.6 mg, 42 mmol) were added. The mixture was

stirred at room temperature for 1 h. The solvent was

removed in vacuo, and the resulting oil was dissolved in

CH₂Cl₂(2 mL) and hexane was added until all the pro-

duct had precipitated. 14 was isolated as a light yellow

(CH₂-NH), 44.1 (CH₂-NH), 47.7 (CH₂-N), 51.0 (CH-

NH), 51.8 (CH-NH), 52.6 (CH₃-0CO), 60.2 (CH-N),

80.3 (Cq), 119.5 (HC), 123.8 (HC), 124.2 (HC),

124.4 (HC), 131.3 (Cq), 135.0 (Cq), 135.5 (Cq),

140.1 (Cq), 156.2 (CO urethane), 168.8 (CO),

170.0 (CO), 170.1 (CO), 170.7 (CO), 171.2 (CO),

200.8 (CO thio ester); Anal. calcd for C₅₇H₉₇N₅O₉S₂

(710.96): C, 64.55; H, 9.12; N, 6.60. Found: C, 64.53; H,

8.93; N, 6.94.

Bis-(N-tert-butyloxycarbonyl-glycyl)-^L-cystine bis(allyl

ester) (17). 1-Hydroxybenzotriazole (6.17 g, 45.72 mmol)

and triethylamine (1.59 mL, 11.43 mmol) were added to

a solution of N-tert-butyloxycarbonyl glycine 5 (2 g,

11.43 mmol) and cystine allyl ester hydro-p-toluene-

sulfonate 9 (3.8 g, 5.71 mmol) in CH₂Cl₂(100 mL). After

stirring for 5 min at room temperature diisopro-

pylcarbodiimide (1.95 mL, 12.57 mmol) was added. The

mixture was allowed to stir for 12 h at room tempera-

ture. The solution was washed with 1 N hydrochloric

acid (100 mL), with aqueous (5%) sodium bicarbonate

(100 mL), and with water (2Â100 mL). The organic

layer was dried over magnesium sulfate, ®ltered and the

solvent was evaporated in vacuo. The crude product

was puri®ed by column chromatography on silica gel

(hexane/ethyl acetate, 1/3 v/v) to aord 17 (2.68 g, 74%)

as a colorless viscous oil: R_f=0.5 (hexane/ethyl acetate,

viscous oil (104.7 mg, 59% for 2 steps): R_f=0.35 (ethyl

ꢀ

acetate); [a]²_d²30 (c 1.57, MeOH); H NMR (MeOD,

1

400 MHz) d 0.83 (t, 3H, J=7.0 Hz, CH₃Pal), 1.21 (s br,

24H, CH₂Pal), 1.42 (s, 9H, CH₃Boc), 1.51±1.63 (m,

2H, CH₂-Me Pal), 1.85±2.21 (m, 4H, 2 CH₂Pro), 2.48

(t, 2H, J=7.4 Hz, CH₂-CO Pal), 2.72±2.80 (dd, 1H,

J=14.0 Hz, J=6.2 Hz, H(H)C-S), 3.34±3.42 (dd, 1H,

J=14.0 Hz, J=4.5 Hz, H(H)C-S), 3.64±3.75 (m, 2H,

CH₂-N Pro), 3.77±3.93 (m, 4H, 2 CH₂-NH), 4.28 (m,

1H, CH-N Pro), 4.76 (m, 1H, CH-NH); ¹³C NMR

(MeOD, 100.5 MHz) d 14.2 (CH₃Pal), 22.7 (CH₂Pal),

25.5 (CH₂Pal), 28.3 (CH₃Boc), 29.0±31.5 (12 CH₂

Pal+2 CH₂Pro), 32.3 (CH₂-S), 42.6 (CH₂-NH), 44.0

(CH₂-NH), 48.3 (CH₂-N), 52.6 (CH-NH), 61.4 (CH-N),

80.1 (Cq), 158.8 (CO urethane), 168.8 (CO), 170.0

(CO), 170.1 (CO), 170.7 (CO), 200.6 (CO thio

ester).

1/2 v/v); [a]_d²²44^ꢀ(c 1.2, CH₂Cl₂); H NMR (CDCl₃,

1

400 MHz) d 1.46 (s, 18H, CH₃Boc), 3.20 (d, 4H,

J=5.0 Hz, CH₂-NH), 3.80±4.00 (m, 4H, CH₂-S), 4.60

(d, 4H, J=6.0 Hz, CH₂-O), 4.90 (m, 2H, CH-NH),

5.20±5.40 (m, 4H, H₂CC), 5.70 (m br, 2H, NH ure-

thane), 5.80±6.00 (m, 2H, HCC), 7.30 (m br, 2H,

NH); ¹³C NMR (CDCl₃, 125.7 MHz) d 28.0 (CH₃Boc),

41.0(CH₂-S), 45.3 (CH₂-NH), 51.8 (CH-NH), 67.1

(CH₂-O), 81.2 (Cq), 119.0 (H₂CC), 132.4 (HCC),

157.1 (CO urethane), 169.7 (CO), 170.0 (CO);

Anal. calcd for C₂₆H₄₂N₄O₁₀S₂(634.23): C, 49.19; H,

6.67; N, 8.82. Found: C, 49.16; H, 6.69; N, 8.77.

N-tert-Butyloxycarbonyl-glycyl-glycyl-(S-palmitoyl)-^L-

cysteyl-^L-prolyl-(S-geranylgeranyl)-L-cysteine methyl ester

(16). Carboxylic acid 14 (33 mg, 49 mmol) and HOBt

(13.3 mg, 98 mmol) were dissolved in CH₂Cl₂(5 mL). (S-

geranylgeranyl)-l-cysteine methyl ester 15 (19.3 mg,

49 mmol) and 5 min later N-(3-dimethylaminopropyl)-

N⁰-ethylcarbodiimide hydrochloride (14 mg, 73.5 mmol)

were added. The mixture was allowed to stir for 24 h at

room temperature. The solvent was evaporated in

vacuo, and the residue was puri®ed by column chroma-

tography on silica gel (ethyl acetate). 16 was obtained

as colorless oil (38 mg, 76%): R_f=0.25 (ethyl acetate);

N-tert-Butyloxycarbonyl-glycyl-(S-palmitoyl)-^L-cysteine

allyl ester (18). Triethylamine (0.77 mL, 5.51 mmol) and

1,4-dl-dithiothreitol (2.12 g, 13.79 mmol) were added

under argon atmosphere to a solution of 17 (1.75 g,

2.75 mmol) in CH₂Cl₂(120 mL). After stirring for 2 h at

room temperature, the solution was washed with 1 N

hydrochloric acid (100 mL), with water (2Â100 mL),

and dried over sodium sulfate. The solution was ®ltered

and cooled to 0 ^ꢀC before adding triethylamine

(0.77 mL, 5.51 mmol) and palmitoyl chloride (4.18 mL,

13.79 mmol). The mixture was stirred for 2 h at room

temperature. After ®ltration and evaporation of CH₂Cl₂

in vacuo, a crude oil was obtained, which was puri®ed

by column chromatography (hexane/ethyl acetate, 1/1

v/v). Compound 18 was isolated as a white solid (2.15 g,

72%): R_f=0.25 (hexane/ethyl acetate, 5/2 v/v); [a]_d²²

[a]²_d²

78^ꢀ(c 1.90, CH₂Cl₂); ¹H NMR (CDCl₃,

500 MHz) d 0.84 (t, 3H, J=7.0 Hz, CH₃Pal), 1.28 (s br,

24H, CH₂Pal), 1.42 (s, 9H, CH₃Boc), 1.55 (m, 2H,

CH₂-Me Pal), 1.58 (s, 9H, 3 CH₃GerGer), 1.67 (s, 3H,

CH₃GerGer), 1.69 (s, 3H, CH₃GerGer), 1.82±2.13 (m,

15H, 6 CH₂GerGer, H(H)C Pro+CH₂Pro), 2.29±2.34

(m, 1H, H(H)C Pro), 2.55 (t, 2H, J=7.3 Hz, CH₂-CO

Pal), 2.72±2.80 (dd, 1H, J=14.0 Hz, J=6.1 Hz, H(H)C-

S), 2.91±3.22 (m, 4H, CH₂-S Cys, CH₂-S GerGer), 3.27±

3.34 (dd, 1H, J=14.0 Hz, J=4.3 Hz, H(H)C-S), 3.64±

3.77 (m, 2H, CH₂Pro), 3.72 (s, 3H, CH₃-OCO), 3.81±

3.93 (m, 4H, 2 CH₂-NH), 4.05±4.13 (m, 1H, CH-N Pro),

4.68 (m, 1H, CH-NH), 4.86 (m, 1H, CH-NH), 5.08 (m,

3H, 3 HC); 5.17 (t, 1H, J=7.0 Hz, HC), 5.24 (s br,

1H, NH urethane), 7.04 (m, 1H, NH), 7.32 (m, 1H,

NH), 7.41 (m, 1H, NH); ¹³C NMR (CDCl₃, 125.7 MHz)

d 14.2 (CH₃Pal), 16.0 (CH₃GerGer), 16.1 (CH₃Ger-

Ger), 16.2 (CH₃GerGer), 17.7 (CH₃GerGer), 22.7

(CH₂Pal), 25.5 (CH₂Pal), 25.7 (CH₃GerGer), 26.6

(CH₂GerGer), 26.7 (CH₂GerGer), 26.8 (CH₂GerGer),

28.4 (CH₃Boc), 29.0±33.2 (12 CH₂Pal, 2 CH₂Pro, 3

CH₂GerGer+CH₂-S Pal), 39.8 (2 CH₂-S GerGer), 42.7

14^ꢀ(c 1.5, CH₂Cl₂); H NMR (CDCl₃, 400 MHz) d

1

0.90 (t, 3H, J=7.0 Hz, CH₃Pal), 1.25 (s br, 24H, CH₂

Pal), 1.47 (s, 9H, CH₃Boc), 1.65 (m, 2H, CH₂-Me Pal),

2.55 (t, 2H, J=7.5 Hz, CH₂-CO Pal), 3.35 (dd, 1H,

J=14.0 Hz, J=6.0 Hz, HC(H)-S), 3.43 (dd, 1H,

J=14.0 Hz, J=4.5 Hz, HC(H)-S), 3.75±3.85 (m, 2H,

CH₂-NH), 4.65 (m, 2H, CH₂-O), 4.80 (m, 1H, CH-NH),

756

T. Schmittberger, H. Waldmann / Bioorg. Med. Chem. 7 (1999) 749±762

5.20 (m br, 1H, NH urethane) 5.25±5.35 (m, 2H,

H₂CC), 5.85±5.95 (m, 1H, HCC), 6.95 (m br, 1H,

NH); ¹³C NMR (CDCl₃, 125.7 MHz) d 14.1 (CH₃Pal),

22.7 (CH₂Pal), 25.5 (CH₂Pal), 28.3 (CH₃Boc), 28.9±

29.6 (CH₂Pal), 30.4 (CH₂Pal), 31.9 (CH₂-S), 44.0

(CH₂-NH), 52.1 (CH-NH), 66.5 (CH₂-O), 80.1 (Cq),

119.0 (H₂CC), 131.3 (HCC), 155.9 (CO urethane),

169.5 (CO), 169.6 (CO), 198.8 (CO thio ester);

Anal. calcd for C₂₉H₅₂N₂O₆S (556.81): C, 62.56; H,

9.41; N, 5.03. Found: C, 62.52; H, 9.18; N, 5.18.

3H, 3ÂCH-NH), 4.91 (s br, 1H, NH urethane), 5.13±

5.21 (dd, 1H, J=10.3 Hz, J=1.5 Hz, H(H)CC), 5.27±

5.35 (dd, 1H, J=17.0 Hz, J=1.5 Hz, H(H)C=C), 5.80±

5.97 (m, 1H, HCH), 7.41 (s br, 1H, NH), 7.95 (s br,

2H, 2ÂNH), 8.26 (s br, 1H, NH); ¹³C NMR (CDCl₃,

62.8 MHz) d 14.1 (CH₃Pal), 15.3 (SCH₃), 21.9 (CH₃

Leu), 22.7 (CH₂Pal), 22.9 (CH₃Leu), 23.5 (CH(Me)₂),

25.6 (CH₂Pal), 28.4 (CH₃Boc), 29.0±30.2 (CH₂Pal),

30.4 (CH₂Met), 31.9 (H₂C-S Met), 41.2 (CH₂Leu), 43.1

(H₂C-NH), 44.0 (H₂C-NH), 50.7 (HC-NH), 52.7 (HC-

NH), 53.2 (HC-NH), 65.8 (H₂C-O), 80.0 (Cq), 118.6

(H₂CC), 131.7 (HCC), 156.4 (CO urethane),

168.8 (CO), 169.7 (CO), 170.4 (CO), 171.2 (CO),

172.7 (CO), 199.6 (CO thio ester); Anal. calcd for

C₄₂H₇₅N₅O₉S₂(858.21): C, 58.78; H, 8.81; N, 8.16;

Found: C, 58.79; H, 8.92; N, 8.03.

N-tert-Butyloxycarbonyl-glycyl-(S-palmitoyl)-^L-cysteine

(19). Tetrakis(triphenylphosphine) palladium (2 mg)

was added under argon atmosphere to a solution of 18

(170 mg, 0.305 mmol) in THF (5 mL). 5 min later mor-

pholine 3 (30 mL, 0.340 mmol) was added. The resulting

mixture was stirred at room temperature for 1 h. The

solvent was removed in vacuo, the resulting oil was dis-

solved in CH₂Cl₂(2 mL) and hexane was added until all

the product had precipitated. 19 was isolated as a yellow

semi-cristalline product (140 mg, 89%) and used with-

N-tert-Butyloxycarbonyl-glycyl-(S-palmitoyl)-^L-cysteyl-L-

methionyl-glycyl-^L-leucine (21). Pentapeptide ester 20

(70 mg, 81.1 mmol) was dissolved, under argon atmo-

sphere, in THF (2 mL). Tetrakis(triphenylphosphine)

palladium (1 mg) and 5 min later morpholine 3 (10.7 mL,

121 mmol) were added. The mixture was allowed to stir

for 3 h at room temperature. The solvent was removed

in vacuo, and the residue was puri®ed by column

chromatography on silica gel (ethyl acetate/MeOH,

7/3+1% acetic acid v/v). 21 was isolated as a light yellow

viscous oil (56.7 mg, 86%): [a]²_d²33^ꢀ(c 1.20, MeOH);

¹H NMR (MeOD, 250 MHz) d 0.89 (t, 3H, J=7.1 Hz,

CH₃Pal), 0.91 (d, 6H, J=4.9 Hz, CH₃Leu), 1.21 (s br,

24H, CH₂Pal), 1.44 (s, 9H, CH₃Boc), 1.48±1.73 (m,

5H, CH Leu, CH₂Leu, CH₂-Me Pal), 1.97±2.25 (m, 2H,

CH₂Met), 2.08 (s, 3H, CH₃Met), 2.50±2.58 (m, 4H,

H₂C-S Met, H2C-CO), 3.22±3.31 (m, 2H, H₂C-S

Cys), 3.85±4.23 (m, 4H, 2ÂH₂C-NH), 4.66 (s br, 2H,

H₂C-O), 4.50±4.72 (m, 3H, 3ÂCH-NH).

out further puri®cation: R_f=0.15 (ethyl acetate/MeOH,

ꢀ

10/1 v/v); [a]_d²²5 (c 1.0, CH₂Cl₂); H NMR (CDCl₃,

1

250 MHz) d 0.90 (t, 3H, J=6.9 Hz, CH₃Pal), 1.26 (s br,

24H, CH₂Pal), 1.49 (s, 9H, CH₃Boc), 1.60±1.73 (m,

2H, CH₂-Me Pal), 2.58 (t, 2H, J=7.5 Hz, CH₂-CO

Pal), 3.22±3.38 (m, 1H, HC(H)-S), 3.42±3.58 (m, 1H,

HC(H)-S), 3.72±3.84 (m, 1H, HC(H)-NH), 3.90±4.03

(m, 1H, HC(H)-NH), 4.70±4.88 (m, 1H, CH-NH), 5.45

(NH urethane), 7.10 (m br, 1H, NH); ¹³C NMR

(CDCl₃, 62.8 MHz) d 14.2 (CH₃Pal), 22.2 (CH₂Pal),

25.5 (CH₂Pal), 28.1 (CH₃Boc), 28.5±30.2 (CH₂Pal),

30.4 (CH₂Pal), 32.1 (CH₂-S), 44.8 (CH₂-NH), 53.1

(CH-NH), 80.0 (Cq), 157.0 (CO urethane), 169.2

(CO), 169.8 (CO), 199.1 (CO thio ester); Anal.

calcd for C₂₆H₄₈N₂O₆S (516.72): C, 60.43; H, 9.36; N,

5.42. Found: C, 60.22; H, 9.21; N, 5.18.

N-tert-Butyloxycarbonyl-glycyl-(S-palmitoyl)-^L-cysteyl-L-

methionyl-glycyl-^L-leucyl-L-proline allyl ester (22). 1-

Hydroxybenzotriazole (19.8 mg, 14.61 mmol) and trie-

thylamine (10.2 mL, 7.34 mmol) were added to a suspen-

sion of 21 (60 mg, 7.34 mmol) and proline allyl ester-

hydro-p-toluenesulfonate 13 (24.1 mg, 7.34 mmol) in

CH₂Cl₂(3 mL). After stirring for 5 min at room tem-

perature, N-(3-dimethylaminopropyl)-N⁰-ethylcarbodi-

imide hydrochloride (21 mg, 10.95 mmol) was added. The

mixture was allowed to stir for 12 h at room tempera-

ture. Then it was washed with 1 N hydrochloric acid

(2 mL), with aqueous (5%) sodium bicarbonate (2 mL),

and ®nally with water (2Â2 mL). The organic layer was

dried over magnesium sulfate, ®ltered, and the solvent

was evaporated in vacuo. The resulting oil was puri®ed

by column chromatography on silica gel (ethyl acetate)

to aord 22 (58.4 mg; 83%) as a colorless oil: R_f=0.70

N-tert-Butyloxycarbonyl-glycyl-(S-palmitoyl)-^L-cysteyl-

L-methionyl-glycyl-L-leucine allyl ester (20). 1-Hydroxy-

benzotriazole (52.1 mg, 0.386 mol) and triethylamine

(40 mL, 0.283 mmol) were added to a solution of tripep-

tide 29a (91.6 mg, 0.193 mmol) and dipeptide 19

(100 mg, 0.193 mmol) in CH₂Cl₂(5 mL) After stirring

for 5 min at room temperature, N-(3-dimethylamino-

propyl)-N⁰-ethylcarbodiimide hydrochloride (55 mg,

0.290 mmol) was added. The mixture was allowed to stir

for 12 h at room temperature, and then was washed with

1 N hydrochloric acid (5 mL), with aqueous (5%)

sodium bicarbonate (5 mL), and ®nally with water

(2Â5 mL). The organic layer was dried over magnesium

sulfate, ®ltered, and the solvent was evaporated in

vacuo. The resulting oil was puri®ed by column chro-

matography on silica gel (ethyl acetate) to aord 20

(116.2 mg, 70%) as a colorless oil: R_f=0.15 (ethyl acet-

(ethyl acetate/MeOH, 8/1 v/v); [a]_d²²

44^ꢀ(c 1.16,

ate); [a]²_d²5.2^ꢀ(c 0.64, CH₂Cl₂); H NMR (CDCl₃,

CH₂Cl₂); H NMR (CDCl₃, 250 MHz) d 0.89 (t, 3H,

J=7.0 Hz, CH₃Pal), 0.92 (d, 3H, J=7.0 Hz, CH₃Leu),

0.97 (d, 3H, J=7.0 Hz, CH₃Leu), 1.22 (s br, 24H, CH₂

Pal), 1.41 (s, 9H, CH₃Boc), 1.49±1.64 (m, 5H, CH Leu,

CH₂Leu, CH₂-Me Pal), 1.99±2.22 (m, 6H, 2ÂCH₂Pro,

CH₂Met), 2.08 (s, 3H, CH₃Met), 2.51±2.58 (m, 4H,

H₂C-S Met, H₂C-CO Pal), 3.17±3.29 (m, 2H, H₂C-S

Cys), 3.90±4.26 (m, 6H, CH₂Pro+2ÂH₂C-NH), 4.64 (s

1

250 MHz) d 0.86 (t, 3H, J=7.1 Hz, CH₃Pal), 0.93 (d,

6H, J=4.9 Hz, CH₃Leu), 1.23 (s br, 24H, CH₂Pal),

1.44 (s, 9H, CH₃Boc), 1.51±1.72 (m, 5H, CH Leu, CH₂

Leu, CH₂-Me Pal), 1.97±2.25 (m, 2H, CH₂Met), 2.08 (s,

3H, CH₃Met), 2.50±2.58 (m, 4H, H₂C-S Met, H₂C-

CO), 3.22±3.31 (m, 2H, H₂C-S Cys), 3.90±4.26 (m,

4H, 2ÂH₂C-NH), 4.66 (s br, 2H, H₂C-O), 4.50±4.72 (m,

T. Schmittberger, H. Waldmann / Bioorg. Med. Chem. 7 (1999) 749±762

757

1

br, 2H, H₂C-O), 4.47±4.63 (m, 4H, 3ÂCH-NH, CH-N),

4.82 (s br, 1H, NH urethane), 5.10±5.22 (dd, 1H,

J=10.3 Hz, J=1.5 Hz, H(H)CC), 5.26±5.35 (dd, 1H,

J=17.0 Hz, J=1.5 Hz, H(H)CC), 5.76±5.95 (m, 1H,

HCH), 7.41 (s br, 1H, NH), 7.95 (s br, 1H, NH), 8.26

(s br, 2H, 2ÂNH); ¹³C NMR (CDCl₃, 62.8 MHz) d 14.0

(CH₃Pal), 15.4 (S-CH₃), 21.8 (CH₃Leu), 22.7 (CH₂

Pal), 23.1 (CH₃Leu), 23.5 (CH(Me)₂), 25.8 (CH₂Pal),

28.4 (CH₃Boc), 29.0±30.2 (CH₂Pal), 30.4±31.0 (CH₂

Met, 2ÂCH₂Pro), 31.9 (H₂C-S Met), 41.0 (CH₂Leu),

43.6 (H₂C-NH), 44.8 (H₂C-NH), 48.0 (CH₂Pro), 51.1

(HC-NH), 52.5 (HC-NH), 53.8 (HC-NH), 54.1 (HC-N),

66.2 (H₂C-O), 80.9 (Cq), 118.3 ((H₂CC), 132.0

(HCC), 157.1 (CO urethane), 169.4 (CO), 169.9

(CO), 170.3 (CO), 170.7 (CO), 171.5 (CO), 172.1

(CO), 199.6 (CO thio ester); Anal. calcd for

C₄₇H₈₂N₆O₁₀S₂(955.33): C, 59.09; H, 8.65; N, 8.79;

Found: C, 59.31; H, 8.81; N, 9.02.

CH₂Cl₂); H NMR (CDCl₃, 400 MHz) d 0.89 (t, 3H,

J=7.0 Hz, CH₃Pal), 0.94 (d, 6H, J=6.6 Hz, 2ÂCH₃

Leu), 1.25 (s br, 24H, CH₂Pal), 1.43 (s, 9H, CH₃Boc),

1.47±1.63 (m, 5H, CH Leu, CH₂Leu, CH₂-Me Pal), 1.59

(s, 6H, 2ÂCH₃Far), 1.66 (s, 3H, CH₃Far), 1.68 (s, 3H,

CH₃Far), 1.99±2.22 (m, 14H, 4ÂCH₂Far, 2ÂCH₂Pro,

CH₂Met), 2.09 (s, 3H, CH₃Met), 2.48±2.61 (m, 4H,

H₂C-S Met, H₂C-CO), 2.68±3.20 (m, 6H, 2ÂH₂C-S

Far, H₂C-S Pal), 3.70±3.90 (m, 4H, 2ÂH₂C-NH), 3.74

(s, 3H, H₃C-OCO), 4.27±4.70 (m, 7H, CH₂Pro,

5ÂCH-NH+CH-N), 5.05 (m, 2H, 2ÂHCC), 5.16 (m,

1H, HCC), 5.41 (s br, 1H, NH urethane), 7.00 (s br,

1H, NH), 7.38 (s br, 2H, 2ÂNH), 7.61 (s br, 1H, NH),

8.00 (s br, 1H, NH); ¹³C NMR (CDCl₃, 100 MHz) d 14.1

(CH₃Pal), 15.6 (S-CH₃), 16.2 (2ÂCH₃Far), 18.0 (CH₃

Far), 22.0 (CH₃Leu), 22.7 (CH₂Pal), 23.3 (CH₃Leu),

23.5 (CH(Me)₂), 25.8 (CH₂Pal), 28.4 (CH₃Boc), 29.0±

30.2 (14 CH₂Pal, CH₃Far, 4ÂCH₃Far), 30.4±31.0

(CH₂Met; 2ÂCH₂Pro), 31.9 (H₂C-S Met), 40.5 (CH₂

Far), 41.2 (CH₂Leu), 43.6 (H₂C-NH), 45.0 (H₂C-NH),

48.0 (CH₂Pro), 51.1 (HC-NH), 51.8 (CH-NH), 52.5

(HC-NH), 53.8 (HC-NH), 54.1 (HC-N), 80.7 (Cq), 118.6

((HCC), 121.6 (HCC), 125.8 (HCC), 132.8 (Cq

Far), 136.1 (Cq Far), 141.0 (Cq Far), 155.9 (CO ure-

thane), 169.7 (CO), 170.0 (CO), 170.3 (CO), 170.7

(CO), 171.1 (CO), 171.5 (CO), 172.1 (CO), 199.6

(CO thio ester); Anal. calcd for C₆₃H₁₀₉N₇O₁₁S₂

(1204.73): C, 62.81; H, 9.11; N, 8.13. Found: C, 63.08;

H, 8.99; N, 9.27.

N-tert-Butyloxycarbonyl-glycyl-(S-palmitoyl)-^L-cysteyl-L-

methionyl-glycyl-^L-leucyl-L-proline (23). Tetrakis(tri-

phenylphosphine) palladium (1 mg) and 1,3-dimethyl

barbituric acid 4 (5 mg, 31.5 mmol) were added under

argon atmosphere to a solution of hexapeptide ester 22

(50 mg, 52.4 mmol) in THF (2 mL). The mixture was

allowed to stir for 2 h at room temperature. The solvent

was removed in vacuo, and the residue was puri®ed by

column chromatography on silica gel (ethyl acetate/

MeOH, 7/3+1% acetic acid v/v). 23 was isolated as a

light yellow viscous oil (40 mg, 84%): R_f=0.60 (ethyl

acetate/MeOH, 8/1 v/v); [a]²_d²37^ꢀ(c 2, MeOH); H

1

Preparation of the tripeptides 29a±d

NMR (MeOD, 250 MHz) d 0.87 (t, 3H, J=7.0 Hz, CH₃

Pal), 0.95 (d, 6H, J=7.1 Hz, 2ÂCH₃Leu), 1.24 (s br,

24H, CH₂Pal), 1.44 (s, 9H, CH₃Boc), 1.46±1.68 (m, 5H,

CH Leu, CH₂Leu, CH₂-Me Pal), 2.01±2.23 (m, 6H,

2ÂCH₂Pro, CH₂Met), 2.09 (s, 3H, CH₃Met), 2.51±

2.58 (m, 4H, H₂C-S Met, H₂C-CO Pal), 3.10±3.21 (m,

2H, H₂C-S Cys), 3.99±4.37 (m, 6H, CH₂Pro, 2ÂH₂C-

NH), 4.54±4.71 (m, 4H, 3ÂCH-NH, CH-N); ¹³C NMR

(MeOD, 62.8 MHz) d 14.1 (CH₃Pal), 15.4 (S-CH₃), 21.9

(CH₃Leu), 22.7 (CH₂Pal), 23.0 (CH₃Leu), 23.4

(CH(Me)₂), 25.8 (CH₂Pal), 28.9 (CH₃Boc), 29.0±30.2

(CH₂Pal), 30.6±31.1 (CH₂Met+2ÂCH₂Pro), 32.2

(H₂C-S Met), 41.1 (CH₂Leu), 43.9 (H₂C-NH), 45.1

(H₂C-NH), 48.2 (CH₂Pro), 51.4 (HC-NH), 52.5 (HC-

NH), 54.0 (HC-NH), 54.7 (HC-N), 80.0 (Cq), 157.6

(CO urethane), 169.4 (CO), 169.7 (CO), 170.0

(CO), 170.7 (CO), 171.9 (CO), 172.6 (CO), 200.2

(CO thio ester).

Synthesis of dipeptide carboxylic acids 26: General pro-

cedure. 1-Hydroxybenzotriazole (4.33 g, 32.10 mmol) and

triethylamine (2.23 mL, 16.05 mmol) were added to a

suspension of glycine allyl ester hydro-p-toluenesulfonate

6 (16.05 mmol) and of a Boc-amino acid (16.05 mmol) in

CH₂Cl₂(80 mL). Diisopropylcarbodiimide (2.73 mL,

17.66 mmol) was added after stirring for 5 min. The

mixture was allowed to stir for 12 h at room tempera-

ture, and then washed with 1 N hydrochloric acid

(50 mL), with aqueous (5%) sodium bicarbonate

(50 mL), and with water (2Â50 mL). The organic layer

was dried over magnesium sulfate, ®ltered, and the sol-

vent was evaporated in vacuo. The crude product was

puri®ed by column chromatography on silica gel (hex-

ane/ethyl acetate, 2/3 v/v). The resulting dipeptide

(8.66 mmol) was dissolved in THF (60 mL). Tetra-

kis(triphenylphosphine) palladium (5 mg) and 5 min

later morpholine 3 (7.54 mL, 86.60 mmol) were added.

The resulting mixture was stirred at room temperature

for 1 h. The solvent was removed in vacuo and the

resulting oil was puri®ed by column chromatography on

silica gel (MeOH/ethyl acetate, 1/4 v/v).

N-tert-Butyloxycarbonyl-glycyl-(S-palmitoyl)-^L-cysteyl-L-

methionyl-glycyl-^L-leucyl-L-prolyl-(S-farnesyl)-L-cysteine

methyl ester (25). Carboxylic acid 23 (40 mg, 43.7 mmol)

and HOBt (11.9 mg, 87 mmol) were dissolved in CH₂Cl₂

(2 mL). (S-farnesyl)-l-cysteine methyl ester 24 (14.8 mg,

43.8 mmol) and 5 min later N-(3-dimethylaminopropyl)-

N⁰-ethylcarbodiimide hydrochloride (12.6 mg, 65.7mmol)

were added. The mixture was allowed to stir for 24 h at

room temperature. The solvent was evaporated in vacuo,

and the residue was directly puri®ed by column chro-

matography on silica gel (ethyl acetate). 25 was isolated

as colorless viscous oil (23.1 mg, 36% for 2 steps): R_f=

0.60 (ethyl acetate/MeOH, 8/1 v/v); [a]²_d²52^ꢀ(c 0.46,

N-tert-Butyloxycarbonyl-^L-methionyl-glycine (26a). 26a

was isolated as a yellow oil (48% for 2 steps). [a]²_d²8^ꢀ

1

(c 1, MeOH); H NMR (MeOD, 250 MHz) d 1.44 (s,

9H, CH₃Boc), 1.81±2.18 (m, 2H, CH₂Met), 2.11 (s,

3H, CH₃Met), 2.57 (m, 2H, H₂C-S), 3.82±4.08 (m, 2H,

H₂C-NH), 4.21±4.33 (m, 1H, HC-NH); ¹³C NMR

(MeOD, 100 MHz) d 15.4 (S-CH₃), 24.8 (CH-(Me)₂),

28.9 (CH₃Boc), 30.5 (CH₂Met), 31.7 (CH₂Met), 42.0

758

T. Schmittberger, H. Waldmann / Bioorg. Med. Chem. 7 (1999) 749±762

(H₂C-NH), 53.6 (HC-NH), 80.6 (Cq), 155.6 (CO ure-

thane), 168.6 (CO).

[a]²_d²

15^ꢀ(c 5.85, CH₂Cl₂); ¹H NMR (CDCl₃,

250 MHz) d 0.92 (d, 6H, J=7.1 Hz, CH₃Leu), 1.38 (d,

3H, J=7.3 Hz, CH₃Ala), 1.43 (s, 9H, CH₃Boc), 1.53±

1.71 (m, 3H, CH Leu; CH₂Leu), 3.84±4.24 (m, 3H, CH-

NH+CH₂-NH), 4.51±4.63 (m, 3H, CH-NH; H₂C-O),

5.20±5.35 (m, 3H, H₂CC; NH urethane), 5.79±5.98 (m,

1H, HCC), 7.15 (s br, 1H, NH), 7.31 (s br, 1H, NH);

¹³C NMR (CDCl₃, 62.8 MHz) d 18.5 (CH₃Ala), 21.6

(CH₂Leu), 22.5 (CH₃Leu), 25.0 (CH(Me)₂), 28.2 (CH₂

Boc), 41.1 (CH₂Leu), 42.0 (CH₂-NH), 50.4 (CH-NH),

50.9 (CH-NH), 66.0 (H₂C-O), 80.2 (Cq), 119.1

(H₂CC), 131.3 (HCC), 155.7 (CO urethane), 169.3

(CO), 171.8 (CO); Anal. calcd for C₁₉H₃₃N₃O₆

(399.49): C, 57.12; H, 8.32; N, 10.51. Found: C, 57.18; H,

8.34; N, 10.50.

N-tert-Butyloxycarbonyl-^L-alanyl-glycine (26b). 26b was

isolated as a light yellow oil (54% for 2 steps): [a]_d²²

+8^ꢀ(c 4.1, MeOH); H NMR (MeOD, 250 MHz) d

1

1.39 (d, 3H, J=7.2 Hz, CH₃Ala), 1.44 (s, 9H, CH₃

Boc), 3.84±4.24 (m, 3H, CH-NH+CH₂-NH); ¹³C NMR

(MeOD, 62.8 MHz) d 18.5 (CH₃Ala), 28.2 (CH₃Boc),

42.7 (CH₂-NH), 50.8 (CH-NH), 80.2 (Cq), 155.1 (CO

urethane), 169.3 (CO).

N-tert-Butyloxycarbonyl-^L-phenylalanyl-glycine (26c). 26c

was isolated as a light yellow oil (51% for 2 steps): [a]_d²²

10^ꢀ(c 3.24, MeOH); H NMR (MeOD, 250 MHz) d

1

1.41 (s, 9H, CH₃Boc), 2.88±3.12 (m, 2H, H₂C-Ph),

3.83±4.07 (m, 2H, H₂C-NH), 4.44±4.56 (m, 2H, CH

Phe), 7.18±7.34 (m, 5H, HCC ar.); ¹³C NMR (MeOD,

62.8 MHz) d 39.9 (CH₂Phe), 42.8 (CH₂-NH), 56.1 (CH-

NH), 128.6, 129.9, 130.1 (HCC ar.), 135.0 (HCC

ar.), 170.2 (CO), 170.7 (CO).

N-tert-Butyloxycarbonyl-^L-phenylalanyl-glycyl-L-leucine

allyl ester (28c). 28c was obtained as a white solid in

65% yield: R_f=0.25 (hexane/ethyl acetate, 1/3 v/v);

[a]²_d²

5^ꢀ(c 2.60, CH₂Cl₂); ¹H NMR (CDCl₃,

250 MHz) d 0.92 (m, 6H, CH₃Leu), 1.39 (s, 9H, CH₃

Boc), 1.58±1.75 (m, 3H, CH Leu, CH₂Leu), 2.91±3.02

(m, 1H, HC(H)-Ph), 3.10±3.20 (m, 1H, HC(H)-Ph),

3.73±3.88 (m, 1H, HC(H)-NH), 3.99±4.11 (m, 1H,

HC(H)-NH), 4.30±4.39 (m, 1H CH-NH Leu), 4.51±4.62

(m, 3H, CH Phe; H₂C-O), 5.04 (m, 1H, NH urethane),

5.18±5.32 (m, 2H, H₂CC), 5.80±5.95 (m, 1H,

HCC), 6.75 (s br, 1H, NH), 6.95 (s br, 1H, NH),

7.18±7.34 (m, 5H, HCC ar.); ¹³C NMR (CDCl₃,

62.8 MHz) d 21.6 (CH₃Leu), 23.2 (CH₃Leu), 25.2 (HC-

(Me)₂), 39.1 (CH₂Phe), 41.6 (CH₂Leu), 42.4 (CH₂-

NH), 53.1 (CH-NH Leu), 56.0, (CH-NH Phe), 66.3

(H₂C-O), 118.4 (H₂CC), 128.8, 130.1, 130.5 (HCC

ar.), 133.3 (HCC), 135.6 (HCC ar.), 170.2 (CO),

170.7 (CO), 173.7 (CO); Anal. calcd for C₂₅H₃₇

N₃O₆(475.59): C, 63.14; H, 7.84; N, 8.83. Found: C,

63.22; H, 8.01; N, 8.91.

Synthesis of tripeptide allyl esters 28: general procedure.

1-Hydroxybenzotriazole (686.2 mg, 5.08 mmol) and

triethylamine (0.35 mL, 2.54 mmol) were added to a

suspension of amino acid allyl ester hydro-p-toluene-

sulfonate 27 (2.54 mmol) and of Boc-dipeptide 26

(2.54 mmol) in CH₂Cl₂/DMF (10/1, 20 mL). Diisopro-

pylcarbodiimide (0.43 mL, 2.79 mmol) was added 5 min

later. The mixture was allowed to stir for 12 h at room

temperature, washed with 1 N hydrochloric acid

(20 mL), with aqueous (5%) sodium bicarbonate

(20 mL), and with water (2Â20 mL). The organic layer

was dried over magnesium sulfate, ®ltered, and evapo-

rated in vacuo. The crude product was puri®ed by col-

umn chromatography on silica gel (hexane/ethyl

acetate, 1/3 v/v).

N-tert-Butyloxycarbonyl-^L-methionyl-glycyl-L-leucine allyl

ester (28a). 28a was isolated as a colorless viscous oil

in 81% yield: R_f=0.30 (hexane/ethyl acetate, 1/3 v/v);

N-tert-Butyloxycarbonyl-^L-methionyl-glycyl-L-alanine allyl

ester (28d). 28d was isolated as a viscous oil in 59%

yield: R_f=0.30 (hexane/ethyl acetate, 1/3 v/v); [a]_d²²

ꢀ

[a]²_d²

18^ꢀ(c 1.80, CH₂Cl₂); ¹H NMR (CDCl₃,

4 (c 5.30, CH₂Cl₂); H NMR (CDCl₃, 250 MHz) d

1.42 (s br, 12H, CH₃Ala, CH₃Boc), 1.84±2.13 (m, 2H,

CH₂Met), 2.11 (s, 3H, CH₃-S), 2.58 (m, 2H, CH₂-S),

3.82±4.08 (m, 2H, H₂C-NH), 4.21±4.32 (m, 1H, CH

Ala), 4.52±4.66 (m, 3H, CH Met, H₂C-O), 5.20±5.39 (m,

3H, H₂CC, NH urethane), 5.80±5.95 (m, 1H,

HCC), 6.92 (s br, 1H, NH), 7.01 (s br, 1H, NH); ¹³C

NMR (CDCl₃, 62.8 MHz) d 15.0 (S-CH₃), 17.7 (CH₃

Ala), 28.9 (CH₃Boc), 29.9 (CH₂Met), 32.0 (CH₂-S), 42.9

(H₂C-NH), 51.1 (HC-NH), 53.0 (HC-NH), 66.4 (H₂C-

O), 80.4 (Cq), 118.8 (H₂CC), 131.9 (HCC), 155.6

(CO urethane), 169.4 (CO), 170.1 (CO), 170.8

(CO); Anal. calcd for C₁₈H₃₁N₃O₆S (417.52): C, 51.78;

H, 7.48; N, 10.06. Found: C, 51.99; H, 7.76; N, 9.77.

1

250 MHz) d 0.93 (d, 6H, J=7.0 Hz, CH₃Leu), 1.42 (s,

9H, CH₃Boc), 1.54±1.71 (m, 3H, CH Leu, CH₂Leu),

1.81±2.18 (m, 2H, CH₂Met), 2.11 (s, 3H, CH₃Met),

2.57 (m, 2H, H₂C-S), 3.82±4.08 (m, 2H, H₂C-NH),

4.21±4.33 (m, 1H, HC-NH), 4.55±4.64 (m, 3H, HC-NH;

H₂C-O), 5.22±5.28 (dd, 1H, J=10.4 Hz, J=1.5 Hz,

H(H)CC), 5.30±5.39 (dd, 1H, J=17.0 Hz, J=1.5 Hz,

H(H)CC), 5.52 (m, 1H, NH urethane), 5.81±6.00 (m,

1H, HCC), 7.12 (m, 1H, NH), 7.23 (m, 1H, NH); ¹³C

NMR (CDCl₃, 100 MHz) d 15.2 (S-CH₃), 21.7 (CH₃

Leu), 22.7 (CH₃Leu), 24.7 (CH-(Me)₂), 28.2 (CH₃Boc),

30.1 (CH₂Met), 31.5 (CH₂Met), 41.0 (CH₂Leu), 42.0

(H₂C-NH), 50.8 (HC-NH), 53.6 (HC-NH), 65.8 (H₂C-

O), 80.1 (Cq), 118.6 (H₂CC), 131.5 (HCC), 155.6

(CO urethane), 168.6 (CO), 172.2 (CO), 172.4

(CO); Anal. calcd for C₂₁H₃₇N₃O₆S (459.60): C, 54.88;

H, 8.11; N, 9.14. Found: C, 54.96; H, 7.89; N, 9.10.

Synthesis of N-terminally deprotected tripeptides 29: general

procedure. Tri¯uoroacetic acid (2.46 mL, 32.2 mmol)

was added to a solution of 28 (1.61 mmol) in CH₂Cl₂

(10 mL). The mixture was allowed to stir for 1 h at room

temperature, and then toluene (30 mL) was added. After

the evaporation of the solvents in vacuo, the desired

products 29 were obtained as viscous oils.

N-tert-Butyloxycarbonyl-^L-alanyl-glycyl-L-leucine allyl ester

(28b). 28b was obtained as a colorless viscous oil in

77% yield: R_f=0.30 (hexane/ethyl acetate, 1/3 v/v);

T. Schmittberger, H. Waldmann / Bioorg. Med. Chem. 7 (1999) 749±762

759

L-Methionyl-glycyl-L-leucine allyl ester hydrotri¯uoro-

acetate (29a). 29a was isolated a_ꢀs a viscous colorless oil

N-tert-Butyloxycarbonyl-glycyl-(S-palmitoyl)-^L-cysteyl-L-

alanyl-glycyl-^L-leucine allyl ester (30a). The pentapep-

tide 30a was obtained as a colorless viscous oil in 85%

in quantitative yield: [a]²_d²+14 (c 0.78, CH₂Cl₂); H

1

NMR (CDCl₃, 250 MHz): d 0.91 (m, 6H, CH₃Leu),

1.52±1.72 (m, 3H, CH Leu, CH₂Leu), 2.03 (s, 3H, CH₃

Met), 2.11±2.24 (m, 2H, CH₂Met), 2.61 (m, 2H, H₂C-

S), 3.62±3.78 (m, 1H, H(H)C-NH), 4.20±4.58 (m, 3H,

H(H)C-NH+HC-NH+H₂C-O), 5.15±5.35 (m, 2H,

H₂CC), 5.73±5.94 (m, 1H, HCC), 7.14 (m, 1H,

NH), 7.23 (s br, 3H, NH₃); ¹³C NMR (CDCl₃,

100 MHz): d 12.1 (S-CH₃), 21.9 (CH₃Leu), 23.3 (CH₃

Leu), 25.9 (CH-(Me)₂), 29.7 (CH₂Met), 31.9 (CH₂

Met), 41.5 (CH₂Leu), 42.8 (H₂C-NH), 52.3 (HC-NH),

53.6 (HC-NH3), 66.7 (H₂C-O), 97.2 (CF₃), 118.7

(H₂CC), 133.2 (HCC), 170.2 (CO), 170.8

(CO), 173.6 (CO), 175.8 (CO); Anal. calcd for

C₁₈H₃₀F₃N₃O₆S (473.51): C, 45.66; H, 6.38; N, 8.87.

Found: C, 45.29; H, 6.17; N, 8.97.

yield: R_f=0.15 (ethyl acetate); [a]_d²²

2.5^ꢀ(c 4.10,

1

CH₂Cl₂); H NMR (CDCl₃, 250 MHz) d 0.88 (t, 3H,

J=6.8 Hz, CH₃Pal), 0.95 (d, 6H, J=5.2 Hz, CH₃Leu),

1.23 (s br, 24H, 12ÂCH₂Pal), 1.44 (s br, 12H, CH₃Boc,

CH₃Ala), 1.53±1.78 (m, 5H, CH Leu, CH₂Leu, CH₂-

Me Pal), 2.49 (t, 2H, J=7.7 Hz, H₂C-CO Pal), 3.24±

3.42 (m, 2H, H₂C-S Cys), 3.91±4.18 (m, 4H, 2ÂH₂C-

NH), 4.24±4.35 (m, 1H, CH-NH), 4.53±4.90 (m, 4H, 2

CH-NH, H₂C-O), 5.21 (dd, 1H, J=10.1 Hz, J=1.2 Hz,

H(H)CC), 5.32 (dd, 1H, J=17.3 Hz, J=1.2 Hz,

H(H)CC), 5.79±5.98 (m, 1H, HCH), 6.05 (s br, 1H,

NH urethane), 7.54 (s br, 1H, NH), 8.21 (s br, 1H, NH),

8.34 (s br, 1H, NH), 8.42 (s br, 1H, NH); ¹³C NMR

(CDCl₃, 62.8 MHz) d 14.1 (CH₃Pal), 22.0 (CH₃Ala),

22.7 (2ÂCH₃Leu), 22.8 (CH₂Pal), 24.8 (CH-(Me)₂),

25.6 (CH₂Pal), 28.4 (CH₃Boc), 29.0±29.9 (12ÂCH₂

Pal), 31.9 (H₂C-S), 41.2 (CH₂Leu), 43.2 (H₂C-NH),

44.0 (H₂C-NH), 49.3 (HC-NH), 50.7 (HC-NH), 52.8

(HC-NH), 65.7 (H₂C-O), 79.7 (Cq), 118.5 ((H₂CC),

131.7 (HCC), 156.4 (CO urethane), 168.9 (CO),

169.4 (CO), 170.5 (CO), 172.5 (CO), 172.7 (CO),

199.8 (CO thio ester); Anal. calcd for C₄₀H₇₁N₅O₉S

(798.09): C, 60.20; H, 8.96; N, 8.77. Found: C, 60.18; H,

8.76; N, 8.66.

L-Alanyl-glycyl-L-leucine allyl ester hydrotri¯uoroacetate

(29b). 29b was isolated as a viscous colorless oil in

quantitative yield: [a]²_d²+12^ꢀ(c 1.80, MeOH); ¹H

NMR (MeOD, 250 MHz) d 0.92 (d, 3H, J=7.0 Hz, CH₃

Leu), 0.96 (d, 3H, J=7.0 Hz, CH₃Leu), 1.52 (d, 3H,

J=7.6 Hz, CH₃Ala), 1.59±1.77 (m, 3H, CH Leu, CH₂

Leu), 3.91±4.08 (m, 3H, CH-NH, CH₂-NH), 4.40±4.50

(m, 1H, HC-NH), 4.60 (d, 2H, J=5.6 Hz, H₂C-O), 5.22

(d, 1H, J=10.2 Hz, H₂CC), 5.34 (d, 1H, J=17.1 Hz,

H₂CC), 5.85±6.01 (m, 1H, HCC).

N-tert-Butyloxycarbonyl-glycyl-(S-palmitoyl)-^L-cysteyl-L-

phenylalanyl-glycyl-^L-leucine allyl ester (30b). The pen-

tapeptide 30b was obtained as light yellow viscous oil

(yield 78%): R_f=0.15 (ethyl acetate); [a]²_d²+1.8^ꢀ(c

L-Phenylalanyl-glycyl-L-leucine allyl ester hydrotri¯uoro-

acetate (29c). The peptide 29c was obtained as a vis-

cous (light yellow) oil with quantitative yield: [a]_d²²

10^ꢀ(c 1.12, MeOH); ¹H NMR (MeOD, 250 MHz)

d 0.91 (d, 6H, J=7.2 Hz, CH₃Leu), 1.55±1.72 (m,

3H, CH Leu, CH₂Leu), 2.88±3.15 (m, 2H, H₂C-Ph),

3.75±3.82 (m, 1H, H₂C-NH), 4.33±4.40 (m, 1H, HC-

NH Leu), 4.25±4.54 (m, 1H HC-NH Phe), 4.63 (d,

2H, J=5.6 Hz, H₂C-O), 5.18 (d, 1H, J=10.2 Hz,

H(H)CC), 5.31 (d, 1H, J=17.0 Hz, H(H)CC),

5.74±5.9 (m, 1H, HCC), 7.12±7.29 (m, 5H, HCC

ar.)

1

2.10, CH₂Cl₂); H NMR (CDCl₃, 400 MHz) d 0.87 (t,

3H, J=6.9 Hz, CH₃Pal), 0.97 (d, 6H, J=5.2 Hz, CH₃

Leu), 1.22 (s br, 24H, 12ÂCH₂Pal), 1.44 (s , 9H, CH₃

Boc), 1.49±.72 (m, 5H, CH Leu, CH₂Leu, CH₂-Me

Pal), 2.50 (t, 2H, J=7.5 Hz, H₂C-CO Pal), 2.88±2.99

(m, 1H, H(H)C-Ph), 3.09±3.20 (m, 1H, H(H)C-Ph),

3.20±3.41 (m, 2H, H₂C-S Cys), 3.88±4.12 (m, 4H,

2ÂH₂C-NH), 4.29±4.35 (m, 1H, CH-NH), 4.50±4.95 (m,

4H, 2 CH-NH+H₂C-O), 5.09 (s br, 1H, NH urethane),

5.19 (dd, 1H, J=10.4 Hz, J=1.3 Hz, H(H)CC), 5.31

(dd, 1H, J=17.3 Hz, J=1.3 Hz, H(H)CC), 5.80±5.96

(m, 1H, HCH), 7.11±7.28 (m, 5H, HC ar.), 7.35 (s br,

1H, NH), 7.98 (s br, 1H, NH), 8.14 (s br, 1H, NH), 8.28

(s br, 1H, NH); ¹³C NMR (CDCl₃, 62.8 MHz) d 14.2

(CH₃Pal), 22.7 (2ÂCH₃Leu), 22.8 (CH₂Pal), 24.9 (CH-

(Me)2), 25.6 (CH₂Pal), 29.0 (CH₃Boc), 29.0±30.1

(12ÂCH₂Pal), 32.0 (H₂C-S), 39.4 (CH₂Phe), 41.4 (CH₂

Leu), 43.7 (H₂C-NH), 44.2 (H₂C-NH), 50.9 (HC-NH),

53.2 (HC-NH), 56.3 (CH-NH Phe), 65.7 (H₂C-O), 80.3

(Cq), 118.8 (H₂CC), 128.8, 130.4, 130.8 (HCC ar.),

132.1 (HCC), 136.2 (HCC ar.), 156.9 (CO ure-

thane), 168.1 (CO), 169.9 (CO), 171.8 (CO), 172.0

(CO), 172.2 (CO), 200.3 (CO thio ester); Anal.

calcd for C₄₆H₇₅N₅O₉S (873.19): C, 63.20; H, 8.64; N,

8.01. Found: C, 63.48; H, 8.79; N, 7.84.

L-Methionyl-glycyl-L-alanyl allyl ester hydrotri¯uoro-

acetate (29d). 29d was isolated as a viscous (colorless)

ꢀ

oil in quantitative yield: [a]²_d²15 (c 1.76, MeOH); H

1

NMR (CDCl₃, 400 MHz) d 1.40 (d, 3H, J=7.3 Hz, CH₃

Ala), 2.04±2.17 (m, 2H, CH₂Met), 2.11 (s, 3H, CH₃-S),

2.61 (t, 2H, J=7.5 Hz, CH₂-S), 3.30 (m, 2H, H₂C-NH),

4.01 (m, 1H, CH Ala), 4.43 (m, 1H, CH Met), 4.61 (d,

2H, J=5.5 Hz, H₂C-O), 5.22 (d, 1H, J=10.3 Hz,

H(H)CC), 5.32 (d, 1H, J=17.2 Hz, H(H)CC),

5.88±5.97 (m, 1H, HCC); ¹³C NMR (CDCl₃,

100 MHz) d 15.1 (S-CH₃), 17.5 (CH₃Ala), 29.7 (CH₂

Met), 31.8 (CH₂-S), 42.8 (H₂C-NH), 50.3 (HC-NH),

53.5 (HC-NH), 66.7 (H₂C-O), 97.9 (CF₃), 118.6

(H₂CC), 133.1 (HCC), 170.0 (CO), 170.4 (CO),

173.6 (CO).

N-tert-Butyloxycarbonyl-glycyl-(S-palmitoyl)-^L-cysteyl-L-

methionyl-glycyl-^L-alanine allyl ester (30c). 30c was iso-

lated as a viscous oil and in 71% yield: R_f=0.15 (ethyl

acetate); [a]²_d²+2.2^ꢀ(c 4.25, CH₂Cl₂); ¹H NMR

(CDCl₃, 250 MHz) d 0.88 (t, 3H, J=7.1 Hz, CH₃Pal),

Preparation of the pentapeptides 30a±c

Peptides 30 were prepared according to the procedure

given for the synthesis of peptides 28.

760

T. Schmittberger, H. Waldmann / Bioorg. Med. Chem. 7 (1999) 749±762

1.22 (s br, 24H, 12ÂCH₂Pal), 1.45 (s, 9H, CH₃Boc),

1.48 (d, 3H, J=7.2 Hz, CH₃Ala), 1.52±1.58 (m, 2H,

CH₂-Me Pal), 2.02±2.24 (m, 2H, CH₂Met), 2.09 (s, 3H,

CH₃Met), 2.49±2.62 (m, 4H, H₂C-S Met, H2C-CO),

3.21±3.42 (m, 2H, H₂C-S Cys), 3.90±4.20 (m, 4H,

2ÂH₂C-NH), 4.24±4.41 (m, 1H, CH-NH) 4.63 (s br,

2H, H₂C-O), 4.65±4.81 (m, 2H, 2ÂCH-NH), 5.02 (s br,

1H, NH urethane), 5.22 (d, 1H, J=10.3 Hz, H(H)CC),

5.34 (d, 1H, J=17.0 Hz, H(H)CC), 5.83±6.02 (m, 1H,

HCH), 6.23 (s br, 1H, NH), 7.83 (s br, 1H, NH), 8.38

(s br, 1H, NH), 8.60 (s br, 1H, NH); ¹³C NMR (CDCl₃,

62.8 MHz) d 14.1 (CH₃Pal), 15.3 (S-CH₃), 18.4 (CH₃

Ala), 22.7 (CH₂Pal), 25.6 (CH₂Pal), 29.0 (CH₃Boc),

29.3±29.8 (CH₂Pal), 30.6 (CH₂Met), 31.9 (H₂C-S Met),

43.2 (H₂C-NH), 44.0 (H₂C-NH), 48.1 (HC-NH), 48.2

(HC-NH), 52.4 (HC-NH), 66.0 (H₂C-O), 79.5 (Cq),

118.6 ((H₂CC), 131.6 (HCC), 156.2 (CO ure-

thane), 168.4 (CO), 169.6 (CO), 169.9 (CO), 171.1

(CO), 172.6 (CO), 198.7 (CO thio ester); Anal.

calcd for C₃₉H₆₉N₅O₉S₂(816.13): C, 57.39; H, 8.52; N,

8.58. Found: C, 57.29; H, 8.75; N, 8.54.

51.0 (HC-NH), 53.9 (HC-NH), 56.3 (CH-NH Phe), 79.8

(Cq), 128.6, 130.8, 130.9 (HCC ar.), 135.9 (HCC

ar.), 157.1 (CO urethane), 168.9 (CO), 170.0 (CO),

170.3 (CO), 170.7 (CO), 171.7 (CO), 199.9 (CO

thio ester).

N-tert-Butyloxycarbonyl-glycyl-(S-palmitoyl)-^L-cysteyl-L-

methionyl-glycyl-^L-alanine (31c). 31c was isolated as a

light yellow solid (y_ꢀield: 82%): [a]²_d²15^ꢀ(c 0.60,

1

MeOH); mp 170±172 C; H NMR (MeOD, 250 MHz)

d 0.89 (t, 3H, J=7.1 Hz, CH₃Pal), 1.22 (s br, 24H, CH₂

Pal), 1.43 (s, 9H, CH₃Boc), 1.47 (d, 3H, J=7.2 Hz, CH₃

Ala), 1.50±1.57 (m, 2H, CH₂-Me Pal), 1.99±2.17 (m, 2H,

CH₂Met), 2.07 (s, 3H, CH₃Met), 2.49±2.58 (m, 4H,

H₂C-S Met, H₂C-CO), 3.20±3.42 (m, 2H, H₂C-S

Cys), 3.88±4.15 (m, 4H, 2ÂH₂C-NH), 4.20±4.32 (m, 1H,

CH-NH), 4.69±4.84 (m, 2H, 2ÂCH-NH); ¹³C NMR

(MeOD, 62.8 MHz) d 14.4 (CH₃Pal), 15.3 (S-CH₃), 18.2

(CH₃Ala), 23.5 (CH₂Pal), 26.4 (CH₂Pal), 28.7 (CH₃

Boc), 29.8±30.8 (CH₂Pal), 31.0 (CH₂Met), 32.7 (H₂C-S

Met), 43.3 (H₂C-NH), 44.6 (H₂C-NH), 50.0 (HC-NH),

54.1 (HC-NH), 54.7 (HC-NH), 80.8 (Cq), 158.1 (CO

urethane), 169.2 (CO), 170.4 (CO), 172.1 (CO),

172.9 (CO), 173.5 (CO), 200.8 (CO thio ester).

Preparation of C-terminally deprotected pentapeptides

31a±c

Peptide carboxylic acids 31 were prepared according to the

general procedure given for the synthesis of peptide 21.

Preparation of the hexapeptides (32a,b)

Peptides 32a,b were prepared according to the general

procedure given for the synthesis of peptide 22.

N-tert-Butyloxycarbonyl-glycyl-(S-palmitoyl)-^L-cysteyl-L-

alanyl-glycyl-^L-leucine (31a). 31a was isolated as a yel-

low viscous oil (yield 70%): [a]²_d²30^ꢀ(c 1.36, MeOH);

¹H NMR (MeOD, 250 MHz) d 0.88 (t, 3H, J=6.8 Hz,

CH₃Pal), 0.97 (d, 6H, J=5.4 Hz, CH₃Leu), 1.21 (s br,

24H, 12( CH₂Pal), 1.44 (s br, 12H, CH₃Boc, CH₃Ala),

1.50±1.72 (m, 5H, CH Leu, CH₂Leu, CH₂-Me Pal),

2.47 (t, 2H, J=7.7 Hz, H₂C-CO Pal), 3.26±3.41 (m,

2H, H₂C-S Cys), 3.97±4.23 (m, 4H, 2ÂH₂C-NH), 4.20±

4.29 (m, 1H, CH-NH), 4.71±4.90 (m, 2H, 2 CH-NH);

¹³C NMR (CDCl₃, 62.8 MHz) d 14.1 (CH₃Pal), 21.3

(CH₃Leu), 22.4 (CH₃Ala), 22.7 (CH₃Leu), 22.9 (CH₂

Pal), 25.0 (CH-(Me)₂), 25.8 (CH₂Pal), 28.8 (CH₃Boc),

28.9±30.3 (12ÂCH₂Pal), 32.2 (H₂C-S), 41.4 (CH₂Leu),

43.9 (H₂C-NH), 44.4 (H₂C-NH), 50.6 (HC-NH), 50.9

(HC-NH), 53.2 (HC-NH), 80.9 (Cq), 156.0 (CO ure-

thane), 168.9 (CO), 169.4 (CO), 171.5 (CO), 172.8

(CO), 173.3 (CO), 200.1 (CO thio ester).

N-tert-Butyloxycarbonyl-glycyl-(S-palmitoyl)-^L-cysteyl-

L-alanyl-glycyl-L-leucine-L-proline allyl ester (32a). 32a

was isolated as a colorless oil (yield 84%): R_f=0.65

(ethyl acetate/MeOH, 8/1 v/v); [a]_d²²

38^ꢀ(c 0.84,

1

CH₂Cl₂); H NMR (CDCl₃, 250 MHz) d 0.87 (t, 3H,

J=7.1 Hz, CH₃Pal), 0.93 (m, 6H, CH₃Leu), 1.22 (s br,

24H, 12ÂCH₂Pal), 1.42 (s, 9H, CH₃Boc), 1.47 (d, 3H,

J=6.8 Hz, CH₃Ala), 1.58±1.76 (m, 5H, CH Leu, CH₂

Leu, CH₂-Me Pal), 1.96±2.21 (m, 4H, 2ÂCH₂Pro), 2.54

(t, 2H, J=7.4 Hz, CH₂-CO Pal), 3.11±3.32 (m, 2H,

CH₂-S Cys), 3.58±3.72 (m, 4H, 2ÂCH₂-NH), 3.82±4.11

(m, 2H, CH₂Pro), 4.23±4.32 (m, 1H, CH-NH), 4.43±

4.52 (m, 2H, CH-NH, CH-N), 4.61 (d, 2H, J=5.5 Hz,

CH₂-O), 4.72±4.83 (m, 1H, CH-NH), 5.21±5.33 (m, 3H,

H₂CC, NH urethane), 5.80±5.97 (m, 1H, HCC),

7.03 (s br, 1H, NH), 7.48 (s br, 2H, 2ÂNH), 7.62 (s br, 1

H, NH), 8.19 (s br, 1H, NH); ¹³C NMR (CDCl₃,

62.8 MHz) d 14.1 (CH₃Pal), 21.6 (CH₃Leu), 22.1 (CH₃

Ala), 22.6 (CH₂Pal), 23.0 (CH₃Leu), 23.5 (CH(Me)₂),

25.7 (CH₂Pal), 28.8 (CH₃Boc), 29.2±30.5 (CH₂Pal),

30.8 (2ÂCH₂Pro), 33.2 (H₂C-S), 40.8 (CH₂Leu), 43.8

(CH₂-NH), 44.4 (CH₂-NH), 48.2 (CH₂Pro), 50.8 (HC-

NH), 51.9 (HC-NH), 52.7 (HC-NH), 53.4 (HC-N), 54.0

(HC-NH), 66.6 (H₂C-O), 80.9 (Cq), 118.5 (H₂CC),

131.7 (HCC), 158.0 (CO urethane), 169.9 (CO),

170.1 (CO), 170.8 (CO), 171.2 (CO), 172.1 (CO),

172.8 (CO), 200.9 (CO thio ester).

N-tert-Butyloxycarbonyl-glycyl-(S-palmitoyl)-^L-cysteyl-L-

phenylalanyl-glycyl-^L-leucine (31b). 31b was isolated as a

viscous oil (yield 87%): [a]²_d²12^ꢀ(c 1.40, MeOH); H

1

NMR (MeOD, 250 MHz) d 0.89 (t, 3H, J=6. Hz, CH₃

Pal), 0.93 (d, 3H, J=5.1 Hz, CH₃Leu), 0.97 (d, 3H,

J=5.4 Hz, CH₃Leu), 1.23 (s br, 24H, 12ÂCH₂Pal),

1.46 (s , 9H, CH₃Boc), 1.51±1.70 (m, 5H, CH Leu, CH₂

Leu, CH₂-Me Pal), 2.50 (t, 2H, J=7.4 Hz, H₂C-CO

Pal), 2.93±3.14 (m, 2H, H₂C-Ph), 3.16±3.39 (m, 2H,

H₂C-S Cys), 3.91±4.14 (m, 4H, 2ÂH₂C-NH), 4.27±4.36

(m, 1H, CH-NH), 4.57±4.78 (m, 2H, 2ÂCH-NH), 7.09±

7.27 (m, 5H, HC ar.); ¹³C NMR (MeOD, 62.8 MHz) d

14.3 (CH₃Pal), 22.2 (CH₃Leu), 22.7 (CH₃Leu), 22.9

(CH₂Pal), 25.0 (CH-(Me)₂), 25.8 (CH₂Pal), 29.1 (CH₃

Boc), 29.2±30.3 (12 CH₂Pal), 32.1 (H₂C-S), 39.6 (CH₂

Phe), 41.9 (CH₂Leu), 43.9 (H₂C-NH), 44.3 (H₂C-NH),

N-tert-Butyloxycarbonyl-glycyl-(S-palmitoyl)-^L-cysteyl-L-

phenylalanyl-glycyl-^L-leucine-L-proline allyl ester (32b).

32b was isolated as a colorless oil (yield 83%):

R_f=0.65 (ethyl acetate/MeOH, 8/1 v/v); [a]²_d²24 (c

1

3.85, CH₂Cl₂); H NMR (CDCl₃, 250 MHz) d 0.91 (t,

T. Schmittberger, H. Waldmann / Bioorg. Med. Chem. 7 (1999) 749±762

761

3H, CH₃Pal), 0.93 (d, 3H, J=5.2 Hz, CH₃Leu), 0.99

(d, 3H, J=5.4 Hz, CH₃Leu), 1.22 (s br, 24H, CH₂Pal),

1.39 (s, 3H, CH₃Boc), 1.52±1.89 (m, 5H, CH Leu, CH₂

Leu, CH₂-Me Pal), 1.93±2.20 (m, 4H 2ÂCH₂Pro), 2.53

(t, 2H, J=7.2 Hz, H₂C-CO Pal), 3.02±3.24 (m, 4H,

H₂C-Ph, H₂C-S Cys), 3.40±3.72 (m, 4H 2ÂCH₂-NH),

3.82±4.04 (m, 2H, CH₂Pro), 4.31±4.50 (m, 3H, 2ÂCH-

NH, CH-N), 4.58 (d, 2H, J=5.4 Hz, H₂C-O), 4.70±4.80

(m, 1H, CH-NH), 5.21±5.36 (m, 3H, H₂C=C, NH ure-

thane), 5.72±5.98 (m, 1H, HCC), 7.07±7.32 (m, 8H,

HC ar.+3ÂNH), 7.40 (s br, 1H NH), 8.31 (s br, 1H,

NH); ¹³C NMR (CDCl₃, 62.8 MHz) d 14.0 (CH₃Pal),

21.9 (CH₃Pal), 22.5 (CH₂Pal), 22.8 (CH₃Leu), 24.7

(CH(Me)₂), 25.8 (CH₂Pal), 29.0 (CH₃Boc), 29.1±30.4

(12 CH₂Pal), 31.1 (CH₂Pro), 31.3 (CH₂Pro), 32.9

(H₂C-S), 39.1 (CH₃Phe), 41.2 (CH₂Leu), 43.2 (CH₂-NH),

44.6 (CH₂-NH), 48.8 (CH₂Pro), 50.2 (HC-NH), 51.5

(HC-NH), 52.7 (HC-NH), 53.6 (HC-NH), 54.8 (HC-

NH), 80.4 (Cq), 118.2 (H₂CC), 128.0, 130.1, 130.5 (HC

ar.), 132.0 (HCC), 136.2 (HC ar.), 158.2 (CO ure-

thane), 170.0 (CO), 170.3 (CO), 170.9 (CO), 171.4

(CO), 172.0 (CO), 200.6 (CO thio ester).

N-tert-Butyloxycarbonyl-glycyl-(S-palmitoyl)-^L-cysteyl-L-

alanyl-glycyl-^L-leucine-L-prolyl-(S-farnesyl)-L-cysteine

methyl ester (35a). 35a was obtained as a colorless oil

(yield 64%): R_f=0.60 (ethyl acetate/MeOH, 8/1 v/v);

[a]²_d²

46^ꢀ(c= 0.43, CH₂Cl₂); ¹H NMR (CDCl₃,

250 MHz) d 0.88 (t, 3H, J=7.0 Hz, CH₃Pal), 0.94

(d, 6H, J=6.5 Hz, 2ÂCH₃Leu), 1.24 (s br, 24H, CH₂

Pal), 1.40±1.45 (s br , 12H, CH₃Boc, CH₃Ala), 1.47±

1.63 (m, 5H, CH Leu, CH₂Leu, CH₂-Me Pal), 1.61 (s,

6H, 2ÂCH₃Far), 1.66 (s, 3H, CH₃Far), 1.69 (s, 3H,

CH₃Far), 1.96±2.23 (m, 12H, 4ÂCH₂Far, 2ÂCH₂

Pro), 2.52 (t, 2H, J=7.3 Hz, H₂C-CO Pal), 2.55±3.22

(m, 4H, 6H, 2ÂH₂C-S Far, H₂C-S Pal), 3.61±3.75 (m,

4H, 2ÂH₂C-NH), 3.73 (s, 3H, H₃C-OCO), 4.11±

4.70 (m, 7H, CH₂Pro, 5ÂCH-NH+CH-N), 5.05 (m,

2H, 2ÂHCC), 5.16 (m, 1H, HCC), 5.43 (s br, 1H,

NH urethane), 6.98 (s br, 1H, NH), 7.48 (s br, 2H,

2ÂNH), 7.64 (s br, 1H, NH), 7.78 (s br, 1H, NH);

¹³C NMR (CDCl₃, 62.8 MHz) d 14.2 (CH₃Pal), 16.5

(2ÂCH₃Far), 18.2 (CH₃Far), 22.0 (CH₃Leu), 22.2

(CH₃Ala), 22.7 (CH₂Pal), 23.5 (CH₃Leu), 23.8

(CH(Me)₂), 25.8 (CH₂Pal), 28.8 (CH₃Boc), 29.1±

30.2 (14 CH₂Pal, CH₃Far, 4ÂCH₂Far), 30.9 (2ÂCH₂

Pro), 40.2 (CH₂Far), 41.6 (CH₂Leu), 43.3 (H₂C-

NH), 44.8 (H₂C-NH), 48.1 (CH₂Pro), 51.6 (HC-NH),

51.9 (CH-NH), 52.7 (HC-NH), 53.2 (HC-NH), 54.0

(HC-N), 79.6 (Cq), 118.9 ((HCC), 121.0 (HCC),

127.2 (HCC), 132.8 (Cq Far), 136.0 (Cq Far), 141.1

(Cq Far), 154.1 (CO urethane), 169.9 (CO),

170.5 (CO), 170.8 (CO), 170.9 (CO), 171.4

(CO), 171.8 (CO), 172.8 (CO), 200.9 (CO thio

ester).

Preparation of C-terminally deprotected hexapeptides (33a,b)

Peptide carboxylic acids 33a,b were prepared according to

the general procedure given for the synthesis of peptide 23.

N-tert-Butyloxycarbonyl-glycyl-(S-palmitoyl)-^L-cysteyl-

L-alanyl-glycyl-L-leucyl-L-proline (33a). 33a was isolated

as a colorless viscous oil (yield 69%): R_f=0.15 (ethyl

acetate/MeOH, 8/1 v/v); [a]²_d²30^ꢀ(c 0.61, CH₂Cl₂);

¹H NMR (CDCl₃, 250 MHz) d 0.88 (t, 3H, J=7.1 Hz,

CH₃Pal), 0.95 (m, 6H, CH₃Leu), 1.23 (s br, 24H, CH₂

Pal), 1.44 (s, 9H, CH₃Boc), 1.49 (d, 3H, J=6.9 Hz, CH₃

Ala), 1.58±1.76 (m, 5H, CH Leu, CH₂Leu, CH₂-Me

Pal), 1.92±2.20 (m, 4H, 2ÂCH₂Pro), 2.53 (t, 2H,

J=7.3 Hz, CH₂-CO Pal), 3.06±3.26 (m, 2H, CH₂-S

Cys), 3.47±3.65 (m, 4H, 2ÂCH₂-NH), 3.84±4.10 (m, 2H,

CH₂Pro), 4.20±4.30 (m, 1H, CH-NH), 4.4±4.59 (m, 2H,

CH-NH+CH-N), 4.69±4.80 (m, 1H, CH-NH), 5.45 (s

br, 1H, NH urethane), 7.09 (s br, 1H, NH), 7.65 ( s br,

3H, 3ÂNH), 7.72 (s br, 1 H, NH).

N-tert-Butyloxycarbonyl-glycyl-(S-palmitoyl)-^L-cysteyl-

L-phenylalanyl-glycyl-L-leucine-L-prolyl-(S-farnesyl)-L-

cysteine methyl ester (35b). 35b was isolated as a light-

yellow oil (yield 53%): R_f=0.55 (ethyl acetate/MeOH,

8/1 v/v); [a]²_d²

44^ꢀ(c 1.41, CH₂Cl₂); ¹H NMR

(CDCl₃, 400 MHz) d 0.90 (t, 3H, J=7.0 Hz, CH₃Pal),

0.96 (m, 6H, 2ÂCH₃Leu), 1.26 (s br, 24H, CH₂Pal),

1.42 (s, 9H, CH₃Boc), 1.49±1.60 (m, 5H, CH Leu, CH₂

Leu, CH₂-Me Pal), 1.64 (s, 6H, 2ÂCH₃Far), 1.67 (s,

3H, CH₃Far), 1.70 (s, 3H, CH₃Far), 1.90±2.18 (m,

12H, 4ÂCH₂Far, 2ÂCH₂Pro), 2.54 (t, 2H, J=7.5 Hz,

H₂C-CO Pal), 2.59±3.27 (m, 8H, 2ÂH₂C-S Far, H₂C-

S Pal, CH₂Phe), 3.74±3.91 (m, 4H, 2ÂH₂C-NH), 3.79

(s, 3H, H₃C-OCO), 4.15±4.80 (m, 7H, CH₂Pro,

5ÂCH-NH, CH-N), 5.04 (m, 2H, 2ÂHCC), 5.18 (m,

1H, HCC), 5.29 (s br, 1H, NH urethane), 6.98 (s br,

1H, NH), 7.03 (s br, 1H, NH), 7.14±7.31 (m, 6H, HC

ar.+NH), 7.44 (s br, 1H, NH), 8.05 (s br, 2H, 2ÂNH);

¹³C NMR (CDCl₃, 100 MHz) d 14.1 (CH₃Pal), 16.6

(2ÂCH₃Far), 18.0 (CH₃Far), 22.3 (CH₃Leu), 22.6

(CH₂Pal), 23.7 (CH₃Leu), 23.8 (CH(Me)₂), 26.0 (CH₂

Pal), 29.1 (CH₃Boc), 29.0±30.0 (14 CH₂Pal, CH₃Far,

4ÂCH₂Far), 30.7 (2ÂCH₂Pro), 39.2 (CH₂Phe), 40.4

(CH₂Far), 41.9 (CH₂Leu), 43.3 (H₂C-NH), 44.0 (H₂C-

NH), 48.1 (CH₂Pro), 51.8 (HC-NH), 52.1 (CH-NH),

52.3 (HC-NH), 52.9 (HC-NH), 54.6 (HC-N), 81.0 (Cq),

118.9 ((HCC), 121.0 (HCC), 127.2 (HCC),

128.1, 130.2, 131.4 (HCC ar.), 133.4 (Cq Far), 136.2

(HCC ar.), 137.1 (Cq Far), 142.6 (Cq Far), 154.0

(CO urethane), 169.2 (CO), 169.9 (CO), 170.2

N-tert-Butyloxycarbonyl-glycyl-(S-palmitoyl)-^L-cysteyl-L-

phenylalanyl-glycyl-^L-leucyl-L-proline (33b). 33c was iso-

lated as a colorless viscous oil (yield 69%): R_f=0.10

(ethyl acetate/MeOH, 8/1 v/v); [a]_d²²

21 (c 1.36,

1

CH₂Cl₂); H NMR (CDCl₃, 250 MHz) d 0.90 (t, 3H,

CH₃Pal), 0.97 (m, 6H, CH₃Leu), 1.23 (s br, 24H, CH₂

Pal), 1.42 (s, 3H, CH₃Boc), 1.50±1.81 (m, 5H, CH Leu,

CH₂Leu, CH₂-Me Pal), 1.90±2.11 (m, 4H 2ÂCH₂Pro),

2.51 (t, 2H, J=7.2 Hz, H₂C-CO Pal), 2.99±3.25 (m,

4H, H₂C-Ph, H₂C-S Cys), 3.43±3.70 (m, 4H, 2ÂCH₂-

NH), 3.87±4.08 (m, 2H, CH₂Pro), 4.22±4.56 (m, 3H,

2ÂCH-NH+CH-N), 4.73±4.85 (m, 1H, CH-NH),

5.21±5.36 (m, 1H, NH urethane), 7.07±7.32 (m, 8H, HC

ar.+3ÂNH), 7.40 (s br, 1H NH), 8.31 (s br, 1H, NH).

Preparation of the heptapeptides (35a±c)

Peptide methyl esters 35a±b were prepared according to

the general procedure given for the synthesis of peptide 25.

762

T. Schmittberger, H. Waldmann / Bioorg. Med. Chem. 7 (1999) 749±762

(CO), 170.5 (CO), 171.7 (CO), 172.1 (CO), 173.2

(CO), 201.0 (CO thio ester).

5. Levitzki, A. Eur. J. Biochem. 1994, 226, 1.

6. Spargaaren, M.; Bischo, J. R.; McCormick, F. Gene Expr.

1995, 4, 345.

7. Hancock, J. F.; Magee, A. I.; Chields, J. E.; Marshall, C. J.

Cell, 1989, 57, 1167.

8. Hall, A. Science 1994, 264, 1413.

9. Okada, T.; Masuda, T.; Shinkai, M.; Kariya, K.; Kataoka,

T. J. Biol. Chem. 1996, 271, 4671.

10. Stokoe, D.; McCormick, F. EMBO J. 1997, 16, 2384.

11. Fernandez-Sarabia, M. J.; Bischo, J. P. Nature 1993,

366, 274.

12. Levitzki, A.; Gazit, A. Science 1995, 267, 1782.

13. Reviews on the relationship between organic synthesis and

biological signal transduction: (a) Waldmann, H.; Hinterding,

K.; Alonso-Diaz, D. Angew. Chem. 1998, 110, 716. (b) Angew.

Chem., Int. Ed. Engl. 1998, 37, 2238. (b) Schmittberger, T.;

Waldmann, H. Synlett 1998, 574. (c) Eisele, F.; Owen, D.;

Waldmann, H. Bioorg. Med. Chem. 1999, 7, 193.

14. Bhatnagar, R. S.; Gordon, J. I. Trends Cell Biol. 1997, 7,

14.

N-t-Butyloxycarbonyl-glycyl-(S-palmitoyl)-^L-cysteyl-L-

methionyl-glycyl-^L-alanyl-L-prolyl-(S-farnesyl)-L-cysteine

methyl ester (35c). 1-Hydroxybenzotriazole (8.7 mg,

6.44 mmol) was added to a suspension of 31c (25 mg,

3.22 mmol) and l-prolyl-(S-farnesyl)-l-cysteine methyl

ester 34²¹(14 mg, 3.22 mmol) in CH₂Cl₂/DMF (10/1,

2 mL). N-(3-dimethylaminopropyl)-N⁰-ethylcarbodii-

mide hydrochloride (9.26 mg, 4.83 mmol) was added

after stirring for 5 min at room temperature. The mix-

ture was allowed to stir for 12 h at room temperature,

and then was washed with 0.5 N hydrochloric acid

(2 mL), with aqueous (5%) sodium bicarbonate (5 mL),

and ®nally with water (2Â5 mL). The organic layer was

dried over magnesium sulfate, ®ltered, and the solvents

were removed in vacuo. The resulting oil was puri®ed by

column chromatography on silica gel (ethyl acetate/

MeOH, 9/1 v/v) to aord 35c (15.3 mg, 41%) as a col-

orless oil: R_f=0.60 (ethyl acetate/MeOH, 8/1 v/v); [a]_d²²

15. Shahinian, S.; Silvius, J. R. Biochemistry 1995, 34, 3813.

16. McLaughlin, S.; Chren, A. Trends Biochem. Sci. 1995, 20,

272.

58^ꢀ(c 0.77, CH₂Cl₂); H NMR (CDCl₃, 400 MHz) d

1

17. (a) Schroder, H.; Leventis, R.; Shahinian, S.; Walton, P.

S.; Silvius, J. R. J. Cell Biol. 1996, 134, 647. (b) Schroder, H.;

Leventis, R.; Rex, S.; Schelhaas, M.; Nagele, E.; Waldmann,

H.; Silvius, J. R. Biochemistry 1997, 36, 13102.

18. Waldmann, H.; Schelhaas, M.; Nagele, E.; Kuhlmann, J.;

Wittinghofer, A.; Schroder, H.; Silvius, J. R. Angew. Chem.

1997, 109, 2334. Angew. Chem. Int. Ed. Engl. 1997, 36, 2238.

19. Ghomashchi, F.; Zhang, X.; Liu, L.; Gelb, M. H. Bio-

chemistry 1995, 34, 11910.

20. Rando, R. Biochim. Biophys. Acta 1996, 1300, 5.

21. Stober, P.; Schelhaas, M.; Nagele, E.; Hagenbuch, P.,

Retey, J.; Waldmann, H. Bioorg. Med. Chem. 1997, 5, 75.

22. Schelhaas, M.; Glomsda, S.; Hansler, M.; Jakubke, H.-D.;

Waldmann, H. Angew. Chem. 1996, 108, 82. Angew. Chem.,

Int. Ed. Engl. 1996, 35, 106.

0.89 (t, 3H, J=7.0 Hz, CH₃Pal), 1.22 (s br, 24H,

12ÂCH₂Pal), 1.45 (s, 9H, CH₃Boc), 1.48 (d, 6H,

J=7.1 Hz, CH₃Ala), 1.52±1.59 (m, 2H, CH₂-Me Pal),

1.61 (s, 6H, 2ÂCH₃Far), 1.66 (s, 3H, CH₃Far), 1.71 (s,

3H, CH₃Far), 2.05±2.31 (m, 14H, 4ÂCH₂Far, 2ÂCH₂

Pro, CH₂Met), 2.11 (s, 3H, CH₃Met), 2.37±2.51 (m,

4H, H₂C-S Met, H₂C-CO), 2.84±3.18 (m, 6H, 2

(H₂C-S Far, H₂C-S Pal), 3.71±3.98 (m, 4H, 2ÂH₂C-

NH), 3.77 (s, 3H, H₃C-OCO), 4.30±4.69 (m, 7H, CH₂

Pro, 5ÂCH-NH, CH-N), 5.06 (m, 2H, 2ÂHCC), 5.14

(m, 1H, HCC), 5.55 (s br, 1H, NH urethane), 7.09 (s

br, 1H, NH), 7.43 (s br, 1H, NH), 7.84 (s br, 2H,

2ÂNH), 8.18 (s br, 1H, NH); ¹³C NMR (CDCl₃,

100 MHz) d 13.9 (CH₃Pal), 15.6 (S-CH₃), 16.6 (2ÂCH₃

Far), 18.1 (CH₃Far), 18.7 (CH₃Ala), 22.7 (CH₂Pal),

23.6 (CH(Me)₂), 25.8 (CH₂Pal), 28.7 (CH₃Boc), 29.1±

30.2 (14 CH₂Pal, CH₃Far, 4ÂCH₃Far), 30.4±31.0

(CH₂Met, 2ÂCH₂Pro), 32.3 (H₂C-S Met), 40.8 (CH₂

Far), 44.1 (H₂C-NH), 44.9 (H₂C-NH), 48.5 (CH₂Pro),

50.8 (HC-NH), 52.0 (CH-NH), 52.9 (HC-NH), 53.8

(HC-NH), 54.4 (HC-N), 79.6 (Cq), 118.0 ((HCC),

121.6 (HCC), 125.8 (HCC), 132.1 (Cq Far), 136.5

(Cq Far), 141.2 (Cq Far), 156.3 (CO urethane), 170.2

(CO), 170.4 (CO), 171.3 (CO), 171.7 (CO), 171.9

(CO), 172.5 (CO), 173.1 (CO), 201.0 (CO thio

ester).

23. Waldmann, H.; Nagele, E. Angew. Chem. 1995, 107, 2425.

Angew. Chem., Int. Ed. Engl. 1995, 34, 259.

24. (a) Nagele, E.; Schelhaas, M.; Kuder, N.; Waldmann, H.

J. Am. Chem. Soc. 1998, 120, 6889. (b) Schelhaas, M.; Nagele,

E.; Kuder, N.; Bader, H. B.; Kuhlmann, J.; Wittinghofer, A.;

Waldmann, H. Chem. Eur. J. 1999, in press.

25. Cotte, A.; Bader, H. B.; Kuhlmann, J.; Waldmann, H.

Chem. Eur. J. 1999, in press.

26. Reviews on protecting group strategies in organic synthesis

in general and in peptide conjugate chemistry in particular:

(a) Kappes, T.; Waldmann, H. Liebigs Ann./Recueil 1997, 803.

(b) Schelhaas, M.; Waldmann, H. Angew. Chem. 1996, 108,

2192; Angew. Chem., Int. Ed. Engl. 1996, 35, 2056.

27. Reviews: Guibe, F. Tetrahedron 1997, 53, 13509; 1998, 54,

2967.

28. Friedrich-Bochnitschek, S.; Waldmann, H.; Kunz, H. J.

Org. Chem. 1989, 54, 751.

29. Review: Kunz, H. Angew. Chem. 1987, 99, 297. Angew.

Chem., Int. Ed. Engl. 1987, 26, 294.

30. Part of this work was published in preliminary form:

Schmittberger, T.; Cotte, A.; Waldmann, H. Chem. Commun.

1998, 937.

31. Kunz, H.; Marz, J. Angew. Chem. 1988, 100, 1424; Angew.

Chem., Int. Ed. Engl. 1988, 27, 1375.

Acknowledgements

This research was supported by the Deutsche For-

schungsgemeinschaft and the Fonds der Chemischen

Industrie. T. S. thanks the Humboldt Foundation for a

research fellowship.

32. Yamane, H, K.; Farnsworth, C. C.; Xie, H.; Howald, W.;

Fung, B. K.-K.; Clarke, S.; Gelb, M. H.; Glomset, J. A. Proc.

Natl. Acad. Sci. USA 1990, 87, 5868.

References and Notes

1. Casey, J. P. Science 1995, 268, 221.

33. Liakopoulou-Kyriakides, M. Phytochemistry 1985, 24,

1593.

34. Petrzik, E.; Kalbacher, H.; Voelter, W. Liebigs Ann.

Chem. 1997, 609.

2. Milligan, G.; Pateri, M.; Magee, A. I. TIBS 1995, 181.

3. Boguski, M. S.; McCormick, F. Nature 1993, 366, 643.

4. Egan, S. E.; Weinberg, R. A. Nature 1993, 365, 781.

Article Doi

Synthesis of the palmitoylated and prenylated C-terminal lipopeptides of the human R- and N-Ras proteins

DOI: 10.1016/S0968-0896(98)00251-X

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