How we drifted into peptide chemistry and where we have arrived at

Tetrahedron 60 (2004) 7455–7506

How we drifted into peptide chemistry and where we

have arrived at

*

ˇ

Dieter Seebach, Thierry Kimmerlin, Radovan Sebesta, Marino A. Campo and Albert K. Beck

Department f u¨ r Chemie und Angewandte Biowissenschaften der Eidgen o¨ ssischen Technischen Hochschule, ETH H o¨ nggerberg,

Wolfgang-Pauli-Strasse 10, CH-8093 Z u¨ rich, Switzerland

Received 16 April 2004

Abstract—The history of peptide chemistry in our group is described. It all started with the cyclic undecapeptide cyclosporin, the

w

immunosuppressive compound, which is commercialised as Sandimmune /Neoral by Sandoz/Novartis, and which has revolutionized

transplant medicine. The discovery that cyclosporin can be deprotonated to a hexalithio derivative, and thus C-alkylated on a sarcosine

moiety, led us into a research project on peptide modiﬁcations. We deﬁned structural prerequisites for the use of peptide enolates and for

electrolytic decarboxylation of peptides. Parallel to these activities, the group was engaged in developing synthetic methodologies aimed at

stereoselective preparations of a-, b-, and g-amino acid derivatives (cf. diastereoselective alkylations, self regeneration of stereogenic

centers, axially chiral enolates). A third avenue into peptide chemistry originated from our investigations on the biopolymer PHB (poly-3-

hydroxybutanoic acid); the question arose ‘what happens upon replacement of chain-bound O by NH in the polyester?’ A brief summary is

given of the results obtained in our ensuing discovery tour of b-peptides built of homologated proteinogenic amino acids. They form

secondary structures with short chain lengths and they have unexpected physiological properties, rendering them candidates for peptidic

3

drugs. The synthesis of b -peptides is straightforward, and in the meantime most of the Fmoc-protected building blocks are commercial. The

2

b -homoamino acids are less readily available. Their preparation and the assembly of a b -eicosapeptide with the twenty proteinogenic side

2

chains are discussed herein. The reasons for the chosen sequence and the strategy of what turned out to be a 159-step synthesis are described.

2

Full experimental details are given for the preparation of the dimeric Fmoc-b hXaa(PG)-b hXaa(PG)-OH building blocks used, for their

2

solid-phase coupling to two b -decapeptide segments, for the thioligation, and for the puriﬁcation, isolation and spectroscopic

characterization of the resulting 20mer. An outlook to future projects in the exciting ﬁeld of b- and g-peptide chemistry and biology is

given.

1

. Introduction

group he was engaged in sulfur-, lithium-, nitroso-, and

nitro-organic chemistry as a synthetic methodologist

(umpolung of reactivity, pool of chiral building blocks).

Later, in Z u¨ rich, the group moved into the areas of

stereoselective transformations, self-regeneration of stereo-

centers, total synthesis of natural products (such as elaio-

phylidin and myxovirescin), structure and mechanisms of

organolithium compounds, use of organotitanium reagents,

TADDOL as a chiral auxiliary system of broad applicability,

all the way to novel crosslinkers for polymerization and

catalysts immobilized on controlled-pore glass.

At the beginning of the senior author’s (D. S.) journey

1

through organic chemistry he would never have expected

to become a peptide chemist towards the end of his

career. The lectures of his mentor in Karlsruhe, Rudolf

Criegee, did not cover this subject at all. If asked, he

would probably have answered, like most organic

chemists at the time, that peptide chemistry is a highly

specialized ﬁeld, and that it is chemically boring to do

nothing but create amide bounds. Thus, D. S. became a

physical organic chemist studying the mechanism of

peroxide decomposition and of cyclobutene ring-opening

reactions. In the postdoctoral work with E. J. Corey in

Cambridge and in the ﬁrst steps into independent research

back in Karlsruhe and later in Giessen with a growing

2

,3

The ﬁrst encounter with peptide chemistry occurred in

one of the senior author’s regular consulting visits at Sandoz

in Basel. The issue was to ﬁnd a more sensitive method of

detection of their immunosuppressive drug cyclosporin A in

plasma. With the experience of our group in organosulfur

4

and -selenium chemistry we treated cyclosporin with

naphtylselenylchloride to induce a selenocyclization of the

Keywords: Cyclosporin; Chiral enolates; Peptide chemistry; b-Peptides.

*

side chain in the unique C -amino acid of the peptide; the

9

product has a much higher extinction coefﬁcient than

Corresponding author. Tel.: þ41-1-632-2990; fax: þ41-1-632-1144;

e-mail address: seebach@org.chem.ethz.ch

doi:10.1016/j.tet.2004.06.043

7456

D. Seebach et al. / Tetrahedron 60 (2004) 7455–7506

7

Figure 1. Formula of cyclosporin A and the THF derivatives formed with strong acid, iodine or C10H SeCl.

Figure 2. Hexalithiated cyclosporin A with a sarcosine enolate moiety that reacts selectively with electrophiles. Different types of amino-acid building blocks

in cyclosporine and rationalization for lack of epimerization and elimination. The sarcosine CH -protons are the least acidic ones!

2

D. Seebach et al. / Tetrahedron 60 (2004) 7455–7506

7457

Figure 3. Li-Enolates of peptides and solubilization of peptides in THF; alkylation of open-chain (a) and of cyclic (b) peptides.

cyclosporin itself, which allowed for HPLC analysis with a

5

detection limit of 5 ng (Fig. 1).

cyclosporin and have non-altered backbone structures.

Si

Those with H -replacement have a different structure and

exhibit different physiological activities.

The cyclic undecapeptide cyclosporin A containing one (R)-

amino-acid and seven N-methyl-amino-acids has actually

been seminal to our group’s entry into the ﬁeld of peptide

chemistry. Returning from another visit to Sandoz in 1983,

where there was a brain-storming session about possible

chemical transformations of cyclosporin, D. S. carried a

bottle of the peptide for an experiment he had proposed,

causing shaking of heads among experts: why not generate a

Li-enolate at the sarcosine residue and introduce side-chains

by reactions with electrophiles? Indeed, treatment of

cyclosporine with as strong bases/nucleophiles as butyl-

lithium (in excess of six equivalents) and addition of typical

electrophiles led to highly selective replacement of either

the Re- or the Si-hydrogen in the sarcosine moiety,

depending on the particular base and conditions

The work on cyclosporin alkylations has triggered a series

6

,11,12

of investigations about peptide enolates,

bilization of peptides in THF by addition of Li salts

(Fig. 3), about direct thionations (CvO ! CvS) of

about solu-

1

0,13,14

1

5

cyclosporin with Lawesson’s reagent, and about cyclo-

1

6

sporin as a Li- and Ca-speciﬁc ionophor.

We started a program entitled ‘chemical modiﬁcations of

peptides’ which led to a number of dissertations in the

group. One line of work was dedicated to the use of

peptides containing amino-malonic-acid derivatives,

1

7

which require only weak bases for alkylations (Fig. 4).

In a quite different investigation we used electrochemical

oxidative decarboxylations of peptides containing up to

ten amino-acid residues, a process leading to modiﬁ-

6

–10

employed; an analysis of how this was possible is

given in Figure 2.

1

8

cations of the C-termini (Fig. 5). Yet another project

was the in situ generation of ketenes for certain peptide

couplings which enabled us to incorporate a single b-

homoamino-acid unit into a larger peptide: activation of

Thus, we have excised a single proton from a peptide of

molecular mass 1200 and replaced it by a side-chain

substituent. The derivatives, in which the diastereotopic Re-

hydrogen had been replaced are immunosuppressive like

the C-terminal CO H-group, reaction with diazomethane,

2

and decomposition of the resulting diazoketone in the

7458

D. Seebach et al. / Tetrahedron 60 (2004) 7455–7506

Figure 4. Peptide alkylation by going from the least to the most acidic CH group and application for peptide modiﬁcation. The process is highly stereoselective

in some cases and is amenable to combinatorial applications.

2

,3

presence of a second peptide with unprotected N-terminus

provides a homologative fragment coupling (Arndt–

enantiopure b -homoamino acids involves formation of

3

the Li -derivative and alkylation of the corresponding b -

2

homoamino acids

review article on b-peptides ). A route to a-branched

1

9

26,27

Eistert sequence of reactions, see Figure 6).

intermediate activate b-homoamino-acid derivative can

The

(for nomenclature see an extensive

2

8

also be trapped with carbohydrates or nucleosides to give

2

chimeric products.

aspartates employs the principle of self-regeneration of

25,29,30

0,21

stereocenters (SRS),

3

and chiral enolates of the achiral

-amino-propanoic acid can be generated from suitable

3

1

b-Homoamino acids had been part of our projects on

synthetic methods for a long time. Following the work on

hydropyrimidines. Formulae of some of these nucleophi-

lic reagents are shown in Figure 7.

2

2,23

dilithiated b-hydroxyesters, for instance malates,

had generated aspartate-enolates

we

as early as 1981. A

general method for the diastereoselective preparation of

2

4,25

We actually conceived the idea of studying b-peptides in the

course of our work on the biopolymer PHB (¼ poly(3-

2

Figure 5. Hofer-Moest Electrolysis of peptides in a protic solvent (H O, MeOH, AcOH) and subsequent Lewis-acid-mediated nucleophilic substitution to N-

terminally modiﬁed peptides.

D. Seebach et al. / Tetrahedron 60 (2004) 7455–7506

7459

Figure 6. Homologative peptide-fragment coupling through a peptidic ketene intermediate. In the absence of an N-methyl-group the actual acylating reagent

may be a di-dehydro-oxazinone. H-b hLeu-OH: (S)-3-amino-5-methyl-hexanoic acid.

3

hydroxybutyrate)): the backbones of cyclic oligo-((R)-3-

hydroxybutanoates) were found to have a propensity to form

of b-homoamino acid residues. Some of the structures are

shown in Figure 9.

(

P)-helical conformations from which a helix could be

modelled, containing chain-bound and carbonyl oxygens at

such a distance and relative position that replacement of the

former by an NH was expected to lead to hydrogen bonding,

Due to the different dimensions, geometries, and polarities

of the b-peptidic structures the biological properties of

b-peptides differ from those of a-peptides in those cases

where exact ﬁtting is mandatory: they do not bind to the

active sites of peptidases and are proteolytically stable.

More surprisingly, they are even metabolically most

2

8,32

and thus stabilization of the helix

(Fig. 8, top).

4

1

Exchange of O by NH in the backbone of a poly(3-

hydroxyalkanoate) renders the same backbone structure as

insertion of a CH -group in each and every amino-acid

4

2,43

stable in mammals, such as rats,

4

in insects, and in

plant-cell cultures, and very slow biodegradation by

environmental microorganisms has been demonstrated in

2

4

residue of a peptide (Fig. 8, bottom). The experimental test

of these ideas led to many chemical, structural, and

biological surprises, a full account of which is given in a

4

5

one case. On the other hand, b-peptides can be used to

mimic a-peptidic hairpin turns, motifs which are often

decisive for so-called ligand–receptor recognitions. This

was demonstrated by the design of N-acyl-b-tetrapeptide

amides with speciﬁc nanomolar binding as agonists at

one of the human receptors for the peptidic hormone

2

8

review article and in ca. 100 papers of our group

since 1996 (see the attached complete list of publications

of D. S.).

As expected, the additional tetrahedral carbon atom in each

amino-acid residue of b-peptides leads to greater structural

variety. There are not just two enantiomeric forms but also

4

6,47

somatostatin (Fig. 10).

2

3

positional isomers (b - and b -homoamino acids), diastereo-

2

One of these peptides was recently shown to be orally

bioavailable, to pass the blood-brain barrier and to

,3

isomers (l- and u-b -homoamino acids with two side

chains), and there can be heteroatoms on the backbone (cf.

4

3

regulate numerous genes in brain tissues. Many other

biological tests have been performed with b-peptides (for

instance inhibition of an intestinal transport protein,

antibiotic and hemolytic activities, binding to DNA and

2

ﬁve b-peptidic helices have been identiﬁed: an 8-,

10/12-, 12-, and 14-helix

the size of the hydrogen-bonded rings within the helix

-halo- or 2-hydroxy-3-amino acids). As a consequence,

2

8,33

34

10-,

(the numbers refer to

3

5

36

37,38

2

8

RNA; see references in a review article ). In recent

4

8

structures). Also, the b-peptidic backbone can be forced to

9

investigations of b-oligoarginine derivatives, it was

shown that these polyelectrolytes enter mammalian cells

in vitro and in vivo to end up in the nucleoli of cell

nuclei (Fig. 11), where they remain located for longer

periods of time (in contrast to corresponding a-oligo-

arginines, which are proteolytically degraded once having

entered cells). There seem to be no toxic effects; the cell

3

adopt a pleated-sheet structure or to form a hairpin turn.

3

4,39

With two exceptions, these structures are seen in

solution with as few as six residues, they can be designed

and found by molecular-modelling programs. Thus, there

are more secondary structures than in the ‘a-world’, but

they can be predicted and constructed from a small number

4

0

Figure 7. Chiral Li-enolates of b-heterosubstituted carboxylic acid esters. Malic acid, b-amino acids, aspartic acid.

7460

D. Seebach et al. / Tetrahedron 60 (2004) 7455–7506

Figure 8. The question ‘what happens upon replacement of O by NH in the modeled helix of PHB?’ (top) is structurally equivalent to the question ‘what

happens upon CH -insertion in the amino acids of a peptide backbone?’ (bottom).

2

5

0,51

culture of human keratinocytes keeps growing in the

presence of the b-oligoarginines.

applying the modiﬁed Evans oxazolidinone DIOZ,

outlined in Figure 12; for details see the original

publications.

as

4

6,48,52–57

Thus we have gone a long way from the ﬁrst experiments

1

4

with cyclosporin A in 1980 to animal experiments with C-

labelled b-peptides in 2003.

Notably, it takes up to 13 steps to prepare some of the acids

2

Fmoc-b hXaa(PG)-OH! Having the b-homoamino acids

available for solid-phase synthesis by the Fmoc strategy

(manual or in a synthesizer), we could make use of all the

methods common in a-peptide synthesis, puriﬁcation,

analysis, structure determination, and modelling (Fig. 13),

with certain adjustments (see discussion in a review

So far, there was no mention made about synthesis of the

b-peptides, which was actually the main occupation of

everybody in the group! At the beginning, we prepared the

3

b -homoamino acids from the corresponding a-amino acids

by Arndt–Eistert homologation ourselves. In the meantime,

2

8

article ).

3

1

8 of the 20 b -homoamino acids with the side chains of the

5

8

proteinogenic a-amino acid analogs are commercial

N-Fmoc- and acid labile side-chain protection); the

For longer-chain b-peptides the thioligation method,

2

3

(

which works equally well for a-, b -, and b -peptide

59

3

49

2

exceptions are b hCys and b hHis. The b -homoamino

acids, on the other hand, have to be prepared enantioselec-

tively. Since our research on b-peptides was focused on

oligomers of homologs of the natural a-amino acids, we

couplings, turned out advantageous. We have recently

addressed the issue, to which chain lengths a b-peptide

might form the 3 -helix: a-peptidic helices in proteins

1

4

are typically only 15–20 residues long, one reason being

a destabilization by the resulting macrodipole which

2

prepared the whole set of the 19 b -homoamino acids

3

2

60

(

b hGly ¼ b hGly!). We use the chiral-auxiliary approach

increases with chain-length. Thus, we have prepared a

D. Seebach et al. / Tetrahedron 60 (2004) 7455–7506

7461

Figure 9. Helix, sheet, and turn structures of b-peptides. Except for the parallel pleated-sheet structure all secondary structures shown can be observed by NMR

spectroscopy of solutions. This Figure has, in part, been reproduced by permission of the Verlag Helvetica Chimica Acta. [Rossi, F.; Lelais, G.; Seebach, D.

Helv. Chim. Acta, 2003, 86, 2653. Etezady-Esfarjani, T.; Hilty, C.; W u¨ thrich, K.; Rueping, M.; Schreiber, J.; Seebach, D. Helv. Chim. Acta, 2002, 85, 1197.]

3

b -eicosapeptide consisting of the 20 different b -homo-

manifold. First of all, we wanted to demonstrate that we

2

6

1

amino acids; the sequence was chosen such that an

amphiphatic helix would result, and stabilization by salt

bridges between (i) and (iþ3)-positions was part of its

design, see the helical-wheel-type presentation in Figure 14.

The CD spectra in methanol and water exhibit an intensive

negative Cotton effect between 210 and 220 nm which we

may consider typical of a 3 -helical secondary structure,

actually can synthesize all the necessary b -homoamino-

acid building blocks with the chiral auxiliary DIOZ. Then,

we decided to ﬁnd ways of avoiding racemization/epimer-

2

ization in b -homoamino-acid coupling, a problem we had

6

3

noticed some time ago. Also, the 3 -helix of short-chain

1

4

2

b -peptides has turned out to be less stable than that of

3

64,65

2

isomeric b -peptides,

so that a comparison of larger b -

1

4

3

however without the usually more intensive short-wave-

3

and b -peptides was important, to ﬁnd out, whether the

former ones fold to other secondary structures. Finally, there

was an atmosphere of sportive ambition in the group about

getting it done!

length maximum seen with short b -peptides. The NMR-

solution structure determination of this 20mer is underway,

and it looks like there is a helix in methanol over the full

6

2

length of the 20 residues.

2

The synthesis of the b -eicosapeptide 1 was designed to be

as safe as possible. To make sure that there would not be

insurmountable problems in the puriﬁcation of the ﬁnal

As the last major project of our group before retirement of

D. S. (with the concomitant necessary reduction of the

research-group size) we joined forces and made essentially

everybody (from advanced lab-course students, through

master-thesis candidates, the last PhD students all the way to

the post-doctoral co-workers) part of a team to synthesize

2

product, we used a convergent synthesis for the b -peptide

(Fig. 15), aware of the fact that there would be more danger

3

of epimerization/racemization than with b -homoamino

3

acids, and remembering that the isolation of the pure b -

eicosapeptide (Fig. 14), assembled in one stroke, had been

2

the all-b -eicosapeptide 1 with the 20 proteinogenic amino-

acid side chains (see below, Fig. 16).

2

8,61

quite cumbersome.

1

8

The reason for embarking on this adventure, which

eventually turned out to be a 159-step synthesis, was

For the choice of the sequence (there are more than 10

possibilities) we applied several different criteria:

7462

D. Seebach et al. / Tetrahedron 60 (2004) 7455–7506

Figure 10. Formula of somatostatin and of a b-tetrapeptide derivative binding to one of the ﬁve human somatostatin receptors.

2

†

The 3 -helix of the eicosapeptide, should it be formed,

1

was supposed to be amphipathic, with stripes of polar and

non-polar side chains on its surface (Fig. 16).

† The choice of b hCys in position 11 is dictated by the

4

2

thioligation, and we put b hAla next to it (position 10) to

reduce steric hindrance in the course of this coupling

process.

Also, the helix should experience salt-bridge stabiliz-

ation, by putting the two pairs of positively and

negatively charged side chains of Arg/Glu and Lys/Asp

in (i)- and (iþ3)-positions, that is, in juxtaposition on the

† Also, the amino acids bearing the side chains of His and

Met have been shown to be incompatible with the types

of reactions (cf. treatment with CH N or with

2

˚

helix at a distance of approximately 5 A (cf. Fig. 9, 3 -

I–CH CN) used for the solid-phase synthesis of peptide

2

thioesters; therefore the corresponding b -homoamino

acids had to be incorporated in the decapeptide 3 with the

1

4

6

8

2

helix, top left).

Next, we considered the well known ‘capping effect’,

6

0

†

2

according to which negative side chains near the positive

and positive side chains near the negative end of a

terminal b hCys residue (Figs. 15 and 16).

2

† The b hPro residue was necessary to be placed in

6

peptidic helix dipol (Fig. 17) have a stabilizing effect,

7

position 20 (i.e., ﬁrst on the Wang resin), because this

amino acid with its secondary piperidine-amino group

does not ﬁt into a 3 -helix; rather it is a hairpin-turn

6

2

also in b-peptides, thus we placed the b hArg in

2

position 3 and the b hAsp in position 17 of the

2

b -eicosapeptide 1.

1

4

69

structural element.

3

Figure 11. Fluorescence microscopy of mouse ﬁbroblast (left), HeLa (center), and human keratinocyte cells (right) after treatment with ﬂuoresceinylated b -

3

oligoarginines consisting of 7, 8 or 10 b hArg residues. This Figure has, in part, been reproduced by permission of the Verlag Helvetica Chimica Acta.

[

Seebach, D.; Namoto, K.; Mahajan, Y. R.; Bindsch a¨ dler, P.; Sustmann, R.; Kirsch, M.; Ryder, N. S.; Weiss, M.; Sauer, M.; Roth, C.; Werner, S.; Beer, H.-D.;

Munding, C. Chem. Biodiversity, 2004, 1, 65.]

D. Seebach et al. / Tetrahedron 60 (2004) 7455–7506

7463

2

Figure 12. Preparation of 19 b -homoamino acid derivatives with the proteinogenic side chains, using enolates derived from the chiral auxiliary DIOZ. For the

2

preparation of Fmoc-(S)b hTrp(Boc)-OH, the classical Evans auxiliary (without the geminal Ph groups) gives better results.

2

Figure 13. The synthesis and analysis of b-peptides from the Fmoc-b - or -b hXaa(PG)-OH is accomplished by the well established methods of a-peptide

3

chemistry (see text books and monographs).

7464

D. Seebach et al. / Tetrahedron 60 (2004) 7455–7506

3

Figure 14. A b -eicosapeptide containing the 20 homologated proteinogenic amino acids. The compound was prepared on solid support and puriﬁed by

preparative HPLC. MS, HPLC of puriﬁed sample, CD (normalized) and NMR spectra, and helical-wheel presentation of an (M)-314-helix, which might be

3

formed by the b -eicosapeptide.

2

†

The b hGly in position 1 was chosen in order to have a

sterically unbiased N-terminus for derivatization.

outlined in the experimental part, where the not

previously described intermediates 13–25, are fully

characterized, including speciﬁc references to their

2

Furthermore, we employed b -dipeptide-fragment coup-

2

ling, to make sure that the two b -decapeptide fragments

2

b hXaa-precursors. The dipeptide-coupling steps are pre-

ceded by numerous protection, deprotection and protective-

group interchange operations. The enantiomer purities of all

and 3 would be easy to purify in case of failure of a

coupling step (a decamer is expected to be separated

more easily from an octamer than from a nonamer); the

2

b hXaa starting materials were checked by HPLC analysis on

chiral columns and/or by NMR spectroscopy of diastereo-

2

b -dipeptide building blocks 4–12 could readily be

isolated in diastereomerically pure form.

The sequence of the dimer segments was, wherever

possible, chosen such that the less epimerization-prone

b -homoamino acid was at the C-, and the more

‘dangerous’ one (Phe, Asp, His, Cys, Tyr, Asn side

chains) at the N-end; in this way, activation of the

carboxylic acid group as active ester would involve

less risk of epimerization.

7

0

meric Pd-complexes. Likewise, the diastereomer purity of

2

the Fmoc-b -dipeptide acids was conﬁrmed by NMR and RP-

†

HPLC analysis before use in the solid-phase coupling steps, to

make sure that no epimerization has occurred during dipeptide

coupling or, else, that any epimer, which might have been

formed, had actually been removed in the chromatographic

puriﬁcation procedure.

2

For the synthesis of b -decapeptide 3 bearing an N-terminal

2

t

The formulae of the suitably protected dipeptide deriva-

tives are shown in Figure 18, and their preparations are

b hCys, by the Fmoc/ Bu solid-phase strategy on Wang

resin, the ﬁrst dipeptide 4 was attached to the resin using the

D. Seebach et al. / Tetrahedron 60 (2004) 7455–7506

7465

7

1

MSNT/MeIm method and the resin loading determined

after treatment with piperidine, 20% in DMF), by

(

measuring the absorbance of the dibenzofulvene-piperidine

adduct at 290 nm (we use the common abbreviations of

7

2

peptide chemistry ). The unreacted OH groups were then

capped’ by acetylation (Ac O and DMAP). Chain

elongation on solid support was performed with HATU

‘

2

and 3 equiv. of the Fmoc-protected b -dipeptides 5–8, and

with piperidine for Fmoc deprotection. After the last

coupling, the peptide was cleaved from the resin and the

side chains deprotected by treatement with CF COOH/

3

EDT/TIS/H O. Finally puriﬁcation by reverse-phase HPLC

2

yielded the b -peptide 3, which was analysed by high-

resolution mass spectrometry (ESI HRMS).

2

The b -peptide 2 bearing a C-terminal thioester was

prepared using the methodology developed by Ingenito

6

8

et al. and based on Kenner’s acylsulfonamide safety-catch

7

3,74

linker.

4

The loading of the resin was achieved with

2

equiv. of the Fmoc-protected b -dipeptide 9, and DIPCDI/

MeIm in DCM/DMF. The peptide was then assembled by

the standard Fmoc protocol (HATU as coupling reagent and

2

Figure 15. Strategy for the synthesis of a b -eicosapeptide through two b -

decapeptides by dimer-fragment solid-phase coupling on sulfonamide (2)

and Wang (3) resin, with subsequent thioligation.

2

Figure 16. Conﬁgurational nomenclature of b-homoamino acids (top), and b -eicosapeptide 1 with central b hAla and b hCys segment, idealized helical-

2

wheel-type presentation of its supposed (M)-314-helix secondary structure (middle), and the two b -decapeptide precursors 2 and 3 for thioligation (bottom).

7466

D. Seebach et al. / Tetrahedron 60 (2004) 7455–7506

3

2

4

Figure 17. Helicity and direction of macrodipole reverse, as we go from a- to b - or b - to g -peptides built of homochiral amino acids. The a- and g-peptidic

helices suffer from destabilizing pole-charge interaction, which is a stabilizing effect in the b-peptidic 314-helix. Furthermore, the resulting macrodipoles

(

increasing with chain lengths) destabilize the helices, which is counteracted by side chains with opposite charge (% near * pole and vice versa). Note that

2

helices built of b hXaa (shown here) and of the enantiomeric building blocks (Figure 12) have opposite helicity. This Figure has, in part, been reproduced by

permission of the Verlag Helvetica Chimica Acta. [Seebach, D.; Schreiber, J. V.; Abele, S.; Daura, X.; van Gunsteren, W. F. Helv. Chim. Acta, 2000, 83, 34.]

3

piperidine for Fmoc deprotection). After the last coupling

step, activation of the safety-catch linker, treatment with

diazomethane followed by a displacement reaction

involving NaSPh/HS(CH ) COOC H led to the still

heterocycle, which is involved with Cys and b hCys as

coupling components (Fig. 20).

We applied this method for the ﬁnal step in the synthesis of

2

5

2

protected decapeptide. Finally, the side chain-protecting

groups were removed in solution by treatment with TFA in

the presence of an appropriate scavenger. In this way we

the b -peptide 1 containing all the b -homoamino acids

with proteinogenic side chains. The ligation was performed

7

9

under standard conditions (aqueous solution, pH 7.5

phosphate buffer and 4% (v/v) PhSH). After 4 h, more than

70% conversion had occurred and the reaction was

essentially complete after 12 h as evident from the

analytical RP-HPLC traces shown in Figure 21.

2

obtained the b -peptide 2. The displacement reaction did

1

ﬁrst not work, even in the presence of LiBr.

0,13,14,75,76

However, heating the reaction mixture at 80 8C overnight,

and deprotection resulted in the formation of the desired

thioester, which was puriﬁed by preparative reverse-phase

HPLC and identiﬁed by high-resolution mass spectrometry.

The crude product 1 was then puriﬁed by preparative

reverse-phase HPLC and characterised by high-resolution

mass spectrometry. The normalized CD spectrum of the

b -eicosapeptide 1 in methanol shows the familiar

negative Cotton effect between 210 and 220 nm with an

intensity similar to that observed with the isomeric

2

CD Spectra of the two b -decapeptides 2 and 3 are shown in

Figure 19.

2

The chemical ligation-methodology, which allows the

coupling of unprotected peptide fragment in aqueous solution,

has made considerable advance in recent years. It offers a new

3

b -eicosapeptide (Fig. 14). However in water the negative

7

7,78

route for the synthesis of larger peptides and proteins.

In

Cotton effect (trough) almost vanishes, and an intensive

positive Cotton effect (peak) appears at shorter wave-

lengths. Similar changes of b-peptidic CD patterns upon

thethioligationreactionthecouplingprocessstartswithatrans

thioesteriﬁcation reaction involving a peptide already bearing

a C-terminal thioester and the sulfhydryl group of a second

peptide bearing an N-terminal Cys. The thioester-linked

intermediate undergoes a subsequent rapid intramolecular

S ! N acyl shift, forming the amide bond at the ligation site.

replacement of MeOH by H O as solvent had been

2

5

8,80–82

observed previously,

and commented with awe

8

1

(‘miraculous’); they may suggest an alteration of the

secondary structures, or unfolding to a ‘totally disor-

dered’ backbone conformation. However, only a full

NMR investigation will be able to elucidate what is going

2

In the case of peptides containing a b -homocysteine, the

intramolecular S ! N acyl shift in the ligation reaction

6

2,82

proceeds through a 6-membered, rather than a 5-membered

on (Fig. 22).

D. Seebach et al. / Tetrahedron 60 (2004) 7455–7506

7467

2

Figure 18. Nine b -dipeptide derivatives 4-12 and the Fmoc-(S)b hTrp(Boc) and b hGly components (N-terminal in 1 and 2) for the solid-phase synthesis of

2

the b -decapeptides 2 and 3 (Figure 16).

2

Figure 19. CD Spectra (normalized) of the two b -decapeptides 2 and 3 in MeOH at þ20 and 210 8C, and in H

2

O. The Cotton effect observed between 215

6

4,65

and 220 nm increases with decreasing temperature. The pattern obtained with 3 we would consider typical of a 314-helix. The shoulder near 225 nm and the

drastic reduction of intensity of the positive Cotton effect near 205 nm seen with the b -decapeptide 2, however, is totally surprising and can not be interpreted

at present. In aqueous pH 7 buffer the b -decapeptide 2 shows a positive Cotton effect at 200 nm albeit with low intensity; the b -decapeptide 3 does not show

2

any signiﬁcant Cotton effect. Compare the CD spectra of the b -eicosapeptide 1 in H O and MeOH in Figure 22 below.

3

Figure 20. Formulae of the cyclic intermediates formed during the S ! N acyl shift in the course of the thioligation reaction involving Cys, b hCys and

2

b hCys.

7468

D. Seebach et al. / Tetrahedron 60 (2004) 7455–7506

2

Figure 21. Analytical-HPLC traces of the ligation reaction between b -decapeptide 2 with a C-terminal thioester group and b -decapeptide 3 with an

2

N-terminal b hCys residue. Samples taken from the reaction mixture at: (a) t ¼ 10 min, (b) t ¼ 2 h, and (c) t ¼ 12 h (chromatographic conditions see

Section 2).

2

,3

39,84

The syntheses of the two b-eicosamers may be taken as a

a-branched b -homoamino-acid residues we

and

2

3

85

demonstration, that any sequence of b - or b -homoamino

acid residues with the proteinogenic side chains can be

assembled. This makes us conﬁdent that we will be able to

construct–by design–b-peptides with tertiary and quatern-

ary structures and, possibly, with catalytic activities. Some

others have observed difﬁculties in the solid-phase

synthesis of corresponding b-peptides. On the other hand,

we are optimistic, as synthetic organic chemists must be ‘by

deﬁnition’, that there will be a solution to any synthetic

problem, if we just try hard enough.

3

evidence for aggregation of long-chain b -peptides (con-

sisting of homologated ‘natural’ a-aminoacid residues) has

Besides construction of more complex architectures with

function, the major goal in the ﬁeld of b-peptides, and

already emerged from concentration-dependent CD

5

8

86

spectra. Also, intramolecular helix–helix interaction has

3

also g-peptides, is the exploitation of their biological,

28

been deduced from CD spectra of a b -peptide with

8

proteinogenic side chains.

pharmacological, and biomedical potential.

Recent

3

experiments with short-chain b-peptides (proteolytically

and metabolitically stable!) have involved structure-

dependent tissue-speciﬁc distributions, gene proﬁling in

brain and lung tissues, afﬁnity to MHC-type-I proteins,

We should, however, not be too sure of our ability to

synthesize any b-peptidic sequence: with the sheet-forming

D. Seebach et al. / Tetrahedron 60 (2004) 7455–7506

7469

2

Figure 22. Formula, mass spectra, HPLC of a puriﬁed sample, CD spectrum (normalized) in MeOH and in H O of b -eicosapeptide 1.

and human-leukocyte-antigen-mediated protection of pig

4

cells against human natural-killer-cell cytotoxicity.

dried in h.v. at 100 8C for 1 h. All other reagents were used

as received from Fluka. TLC: Merck silica gel 60 F₂₅₄

plates; detection with UV or ‘Mo-stain’ solution (25 g

phosphormolybdic acid, 10 g Ce(SO ) ·H O, 60 mL conc.

3

4

2

. Experimental

H SO , 940 mL H O), FC: Fluka silica gel 60 (40–63 mm);

2 4 2

at ca. 0.2 bar. Anal HPLC: Merck HPLC system (LaChrom,

pump type L-7150, UV detector L-7400, Interface D-7000,

HPLC Manager D-7000). Macherey-Nagel C -column

2

.1. General

8

Abbreviations: The ofﬁcial abbreviations of Peptide

7

(Nucleosil 100-5 C

8

250£4 mm); Waters HPLC system

2

Science are used throughout this paper. DMAP

4-(dimethylamino)pyridine), DIPCDI (diisopropylcarbodii-

(pump type 515, data module type 746, tunable absorbance

detector type 484). Chiralcel OD-H column. Prep. HPLC:

Merck HPLC system (LaChrom, pump type L-7150, UV

detector L-7400, Interface D-7000, HPLC Manager D-

(

mide), DIPEA (diisopropylethylamine), EDC(N-(3-dimethyl-

0

ethanedithiol), FC (ﬂash chromatography), FmocOSu

aminopropyl)-N -ethyl-carbodiimide hydrochloride), EDT

(

7000) Macherey-Nagel C column (Nucleosil 100-7 C

8 8

N-(9-Fluorenylmethoxycarbonyloxy)succinimide), HATU

0

(250£21 mm)). Circular dichroism (CD): CD spectra were

recorded on a Jasco J-710 spectropolarimeter from 190 to

250 nm with a Jasco PTC-348 WI Peltier System at 20 8C or

210 8C in 1 mm rectangular cells. The optical system was

0

O-(7-azabenzotriazol-1-yl)-N,N,N ,N -tetramethyluronium

hexaﬂuorophosphate), h.v. (high vacuum, 0.01–0.1 Torr),

-MeIm (1-methylimidazole), NMM (N-methylmorpholine),

1

MSNT (1-(mesitylene-2-sulphonyl)-3-nitro-1H-1,2,4-tria-

zole), TBAF (tetra-n-butylammonium ﬂuoride), TFA (tri-

ﬂuoroacetic acid), TIS (triisopropylsilane), TNBS (2,4,6-

trinitrobenzensulfonic acid). Solvents for chromatography

ﬂushed with N at a ﬂow rate of ca. 10 L/min. Parameters:

2

band width 1.0 nm, resolution 0.2–1 nm, sensitivity

100 mdeg, response 0.5 s, speed 50 nm/min, 5 accumu-

lations. All spectra were corrected for the corresponding

solvent spectrum and normalized. Peptide concentrations

were typically 0.2 mM. The molar ellipticity [u] in

were distilled from Sikkon (anh. CaSO ; Fluka), THF was

4

distilled from Na, CH Cl and NEt from CaH . LiCl was

2

3

2

7470

D. Seebach et al. / Tetrahedron 60 (2004) 7455–7506

2

21

deg·cm ·mol (l in nm). Smoothing was done by Jasco

software. Solvents: MeOH (HPLC grade), aq. Buffer pH

Et O. The aqueous phase was acidiﬁed with 1 N HCl to

2

pH,1 and extracted with AcOEt (3£). The combined

7

(

1

.0: 0.1 M KH PO /0.1 M NaOH. NMR: Bruker AMX 500

H 500 MHz, C 125 MHz), AMX-400 ( H 400 MHz,

00 MHz) and Varian Gemini ( H 300 MHz, C 75 MHz)

organic extracts were dried (MgSO ) and the solvent

4

2

13

4

1

13

C

removed under reduced pressure. The crude product was

puriﬁed by FC.

1

13

chemical shifts d in ppm downﬁeld from internal SiMe₄

0 ppm). Mass Spectra: IonSpec Ultima 4.7 T FT Ion

Cyclotron Resonance (ICR, HR-MALDI, in a 2.5-dihy-

droxybenzoic acid matrix), or Finnigan MAT TSQ 700

(

2.2.4. Fmoc-protection: general procedure 4 (GP4). To a

solution of the N-deprotected dipeptide in 0.15 M Na CO

2

3

(2 equiv.) was added FmocOSu (1.2 equiv.) in acetone

(0.1 M). If necessary, the pH was adjusted to 9–10 with

additional aq. Na CO solution and the mixture stirred at

(

ESI) mass spectrometer; in m/z (% of basis peak). Melting

points: B u¨ chi-510 apparatus; uncorrected. Optical rotations:

Perkin–Elmer 241 polarimeter (10 cm, 1 mL cell, room

temperature). IR: Perkin–Elmer 1600 FT-IR spectropho-

tometer. Elemental analyses were performed by the

Microanalytical Laboratory of the Laboratorium f u¨ r Orga-

nische Chemie, ETH Z u¨ rich.

2

3

25 8C for 4 h. The acetone was carefully removed under

reduced pressure at 30 8C and the resulting mixture diluted

with H O. At this point the pH of the solution was adjusted

2

to 9–10 using 0.6 M aq. Na CO . The aq. mixture was then

2

3

extracted with Et O (2£). The aq. phase was separated,

2

cooled to 0 8C and AcOEt added. With continuous stirring

of the biphasic system at 0 8C, the pH of the aq. phase was

adjusted to 4–5 by slow addition of 10% aq. citric acid. The

org. layer was separated and the aq. layer extracted with

AcOEt (2£). The combined org. layers were washed with

brine, then dried (Na SO ), and concentrated under reduced

2

.2. General procedures

.2.1. Peptide coupling to give dimers 4–12: general

procedure 1 (GP1). The appropriate N-deprotected amino

acid (1 equiv.) was dissolved in CH Cl (0.1 M) and cooled

2

4

in an ice-bath. To the mixture was successively added NMM

3 equiv.) and the N-protected amino acid (1 equiv.). To this

solution either HATU (1.2 equiv.) (GP1a) or EDC

1.2 equiv.) and HOBt (1.2 equiv.) (GP1b) was added and

pressure. The crude product was puriﬁed by FC.

(

2.2.5. Preparation of trimethylsilylethyl esters: general

procedure 5 (GP5). To a solution of the C-unprotected

amino acid (1 equiv.) in CH Cl (0.1 M) at 0 8C under Ar,

(

the mixture allowed to warm up to 25 8C and stirred

overnight. The mixture was then diluted with CH Cl and

2

was added trimethylsilylethanol (1.5 equiv.), DMAP

(0.2 equiv.), and EDC (1.2 equiv.). The resulting mixture

was stirred at 0 8C for 16 h then diluted with AcOEt and the

org. phase washed with 10% Na CO (2£), and brine, then

2

washed with 1 M HCl, 10% aq. K CO and brine solutions.

2

3

The organic phase was dried (MgSO ) and the solvent

4

removed under reduced pressure. The crude dipeptide was

puriﬁed by FC.

2

3

dried (MgSO ), and concentrated under reduced pressure.

4

The crude product was puriﬁed by FC.

2.2.2. Hydrogenolysis of Cbz and Bn-ester groups:

general procedure 2 (GP2). The corresponding substrate

2.2.6. Trimethylsilylethyl ester deprotection: general

procedure 6 (GP6). To a solution of the corresponding

Si-ester (1.0 equiv.) in THF (0.1 M) was added TBAF·3H O

2

(4 equiv.) and the mixture stirred at 25 8C for 1 d. The

reaction mixture was then diluted with AcOEt and the

was dissolved in either MeOH or THF (0.02 M) and ca. 10%

(

w/w) Pd/C (10%) was added. The apparatus was evacuated

and ﬂushed with H (3£), and the solution was stirred under

2

an atmosphere of H for the indicated amount of time

2

(

monitoring by TLC). Subsequent ﬁltration through Celite

organic phase washed with sat. aq. NH Cl, and brine, then

4

and removal of solvent under reduced pressure yielded the

product, which was used in the next step without further

puriﬁcation.

dried (MgSO ), and concentrated under reduced pressure.

4

The crude product was puriﬁed by FC.

2.2.7. Reversed-phase (RP) HPLC analysis and puriﬁ-

2.2.3. Saponiﬁcations: general procedure 3 (GP3). The

appropriate ester (1 equiv.) was dissolved in MeOH/H O

cation. RP-HPLC analysis was performed on a Macherey-

Nagel C column (Nucleosil 100-5 C (250£4 mm)) by

2

8

3

:1 (0.1 M) at 25 8C. To the resulting solution, LiOH·H O

2

using a linear gradient of A (0.1% TFA in H O) and B

2

(

3

2.5 equiv.) was added and the reaction mixture was stirred

h. The mixture was diluted with H O and extracted with

(MeCN) at a ﬂow rate of 1.2 mL/min with UV detection at

220 nm; t in min. RP-HPLC puriﬁcation was performed on

2

R

2

Figure 23. Preparation of Fmoc-(S)b hPhe-(S)b hPro-OH (4).

D. Seebach et al. / Tetrahedron 60 (2004) 7455–7506

7471

a Macherey-Nagel C₈column (Nucleosil 100-5 C₈

250£21 mm)) by using a linear gradient of A and B at a

ﬂow rate of 18 mL/min (Merck HPLC system).

42.0, 42.1, 42.8, 43.1, 43.2, 45.0, 47.0, 65.1, 126.0, 126.1,

127.5, 126.6, 126.7, 128.1, 128.15, 128.22, 128.24, 128.6,

128.8, 137.1, 137.2, 139.2, 139.3, 156.1, 170.9, 171.1,

(

1

74.0, 174.2; MALDI HRMS calcd for C H N O Na

24 28 2 5

þ

C H N O : C 67.91, H 6.65, N 6.60; found: C 67.89, H

6.45, N 6.54.

2

.3. Preparation of Fmoc-protected dipeptides 4–12

(MþNa) : 447.1890, found: 447.1887. Anal. calcd for

2

4 28 2 5

2

.3.1. Cbz-(S)b hPhe-(S)b hPro-OEt (13). Amino acids

87

2

Cbz-(S)b hPhe-OH

2

(1.05 g, 3.35 mmol) and H-

S)b hPro-OEt (0.53 g, 3.35 mmol) were coupled accord-

88

2

(

2.3.3. Fmoc-(S)b hPhe-(S)b hPro-OH (4). Cbz-dipeptide

14 was hydrogenolyzed according to GP2 then Fmoc-

protected according to GP4. The crude peptide was puriﬁed

by FC (AcOEt/hexane/AcOH 10:10:0.1) to give 4 (1.13 g,

ing to GP1a. FC (AcOEt/hexane 1:1) gave 13 (1.44 g, 95%)

as colorless oil; R ¼0.23 (AcOEt/hexane 1:1); [a] ¼þ27.5

f

D

(

c¼0.80, CHCl ); IR (CHCl ) n

3450 (w), 3007 (m),

max

3

2

1

944 (w), 1720 (s), 1626 (s), 1511 (s), 1454 (m), 1139 (m),

1

98%) as a white foam; mp 84–87 8C; R ¼0.35 (AcOEt/

f

085 (m), 1030 (m), 856 (w) cm². H NMR (500 MHz,

1

hexane/AcOH 10:10:1); [a] ¼21.6 (c¼0.73, CHCl ); IR

D

3

DMSO-d ) 13þrotamers d 0.92–1.14 (m, 2H, CH ), 1.17 (t,

(CHCl ) n

3

3450 (w), 3008 (m), 2949 (m), 2862 (w), 1713

max

6

2

J¼6.3 Hz, 3H, CH ), 1.26–1.50 (m, 2H, CH ), 1.66–1.84

(s), 1625 (s), 1514 (s), 1467 (m), 1450 (s), 1181 (m), 1144

3

2

1 1

(

m, 2H, CH ), 2.29 (m, 1H, CH), 2.51–2.90 (m, 4H, CH,

2

(m), 1084 (m), 1008 (m), 990 (w), 856 (w) cm²; H NMR

CH , CHH), 3.06–3.19 (m, 2H, CH N), 3.67 (t, J¼14.4 Hz,

(400 MHz, CD OD) 4þrotamers d 1.02–1.99 (m, 4H,

2

3

1H, NH), 4.03 (m, 2H, OCH ), 4.27 (d, J¼11 Hz, 1H,

CHH), 5.01 (m, 2H, OCH Ph), 7.07–7.49 (m, 10H, arom.);

2£CH ), 2.27–2.93 (m, 4H, 2£CH and CH ), 3.21–3.79

2

(m, 4H, 2£CH ), 4.21 (m, 2H, CH ), 4.30–4.53 (m, 3H,

C NMR

2

1

3

13

C NMR (125 MHz, DMSO-d ) 13þrotamers d: 13.9,

OCH CH), 7.08–7.79 (m, 13H, arom.);

6

2

4

1

3.7, 24.4, 26.5, 26.7, 35.8, 36.3, 40.0, 40.5, 40.6, 41.4,

1.9, 42.2, 42.9, 43.1, 43.2, 45.0, 46.8, 59.8, 59.9, 65.1,

26.1, 127.6, 128.1, 128.2, 128.8, 137.2, 139.3, 156.1,

(100 MHz, CD OD) 4þrotamers d 21.5, 25.35, 25.41,

3

25.7, 26.4, 28.4, 28.6, 37.8, 38.1, 42.39, 42.48, 42.54, 43.3,

43.5, 43.6, 44.1, 44.5, 44.6, 44.8, 44.83, 44.86, 44.93, 45.1,

45.3, 47.4, 67.6, 67.7, 67.8, 67.9, 121.0, 126.0, 126.1, 126.2,

126.3, 127.5, 127.6, 127.7, 128.2, 128.8, 129.3, 129.5,

129.6, 129.7, 130.0, 130.3, 140.3, 140.7, 142.7, 145.4,

158.9, 174.2, 174.3, 174.4, 176.2, 176.6, 176.9; MALDI

71.0, 172.2, 172.4; MALDI HRMS calcd for

þ

C H N O Na (MþNa) : 475.2203, found: 475.2207.

2

6 32 2 5

Anal. calcd for C H N O : C 69.01, H 7.13, N 6.19;

2

6 32 2 5

found: C 69.01, H 7.16, N 5.93 (Fig. 23).

þ

HRMS calcd for C H N O Na (MþNa) : 535.2203;

3

1 32 2 5

2

.3.2. Cbz-(S)b hPhe-(S)b hPro-OH (14). Dipeptide ester

3 (1.34 g, 2.96 mmol) was hydrolyzed according to GP3.

found: 535.2199. Anal. calcd for C H N O : C 72.64, H

31 32 2 5

1

The resulting crude material was crystallized from CHCl₃/

hexane to give 14 (1.02 g, 81%) as white crystals; mp 189–

6.29, N 5.46; found: C 72.71, H 6.43, N 5.24 (Fig. 23).

2

2.3.4. Boc-(S)b hMet-OCH CCl (15). The crude H-

2

3

2

55

1

90 8C;

R ¼0.32

(AcOEt/hexane/AcOH

10:10:1);

3446 (w),

max

(S)b hMet-OH

(2.5 mL) and aq. 1 M NaOH (5 mL) at 5 8C (ice bath). To

(2.42 mmol) was dissolved in H O

2

f

[

3

1

a] ¼þ2.4 (c¼0.15, CHCl ); IR (CHCl ) n

D

3

008 (m), 1715 (s), 1627 (s), 1513 (m), 1454 (m), 1082 (m),

the resulting solution Boc O (0.63 g, 2.90 mmol, 1.2 equiv.)

2

046 (m), 1005 (w), 877 (w) cm²; H NMR (400 MHz,

1

in dioxane (5 mL), was added and the mixture stirred for

30 min at 25 8C. The solution was concentrated to half of its

original volume, cooled again in an ice bath, covered with a

layer of AcOEt and acidiﬁed with a dilute solution of

DMSO-d ) 14þrotamers d 0.91–1.50 (m, 5H, 2£CH and

6

2

CHH), 1.68–1.91 (m, 2H, CH ), 2.21–2.86 (m, 3H, NH,

2

CH, CHH), 3.06–3.19 (m, 2H, CH ), 3.54 (m, 1H, CH),

2

4

CHH), 5.02 (m, 2H, OCH Ph), 7.06–7.39 (m, 10H, arom.),

.20 (d, J¼12.9 Hz, 1H, CHH), 4.43 (d, J¼10.7 Hz, 1H,

KHSO to pH 2–3. The aqueous phase was extracted with

4

AcOEt (2£). The combined organic extracts were washed

2

1

3

1

2.32 (br s, 1H, COOH); C NMR (100 MHz, DMSO-d₆)

4þrotamers d: 23.9, 24.6, 26.8, 26.9, 36.3, 40.6, 40.7, 41.5,

with H O, dried (MgSO ) and the solvent removed under

2

4

1

reduced pressure. The crude product (0.45 g, 1.71 mmol)

2

t

Figure 24. Preparation of Fmoc-(S)b hAsp(O Bu)-(S)b hMet-OH (5).

2

7472

D. Seebach et al. / Tetrahedron 60 (2004) 7455–7506

and DMAP (42 mg, 0.34 mmol, 20 mol%) were dissolved in

CH Cl (20 mL) and Cl CCH OH (0.19 mL, 0.28 g,

1.88 mmol, 1.1 equiv.) was added. The resulting solution

was cooled in an ice bath then EDC (0.39 g, 2.05 mmol,

(ice bath), Zn powder (3.99 g, 61 mmol, 50 equiv.) was

added in portions over 2 h. The reaction mixture was

allowed to warm up to 25 8C and stirred for 3 h. Zn was

2

3

2

removed by ﬁltration and the ﬁltrate diluted with H O and

2

1

2

.2 equiv.) added and the reaction mixture stirred for 18 h at

5 8C. Sat. aq. NH Cl was added and the organic phase

extracted with AcOEt (3£). The combined organic extracts

were dried (MgSO ) and the solvent removed under reduced

4

washed with 0.1 N HCl, 0.1 M K CO and brine then dried

2

pressure. FC (AcOEt/hexane/AcOH 10:10:0.1) of the crude

product yielded 5 (0.65 g, 93%) as a white solid; mp 143–

3

(

crude product was puriﬁed by FC (AcOEt/hexane 3:7) to

MgSO ) and solvent removed under reduced pressure. The

4

144 8C;

R ¼0.37

(AcOEt/hexane/AcOH

10:10:1);

3436 (w),

max

f

give 15 (0.56 g, 59%) as colorless oil; R ¼0.61 (AcOEt/

[a] ¼þ15.9 (c¼0.43, CHCl ); IR (CHCl ) n

f

D

3

hexane 1:1); [a] ¼þ3.1 (c¼0.26, CHCl ); IR (neat) n

3005 (m), 2974 (m), 1720 (s), 1667 (m), 1512 (m), 1450

2

D

3

max

1 1

3

1

344 (w), 3054 (w), 2976 (w), 2140 (m), 1752 (s), 1710 (s),

513 (m), 1445 (m), 1366 (m), 1272 (m), 1250 (m), 1167

(m), 1368 (m), 1154 (m), 1077 (w), 841 (w) cm ; H NMR

t

(300 MHz, CD OD) d 1.43 (s, 9H, Bu), 1.84 (m, 2H,

3

2

1 1

(

s), 789 (m) cm ; H NMR (300 MHz, CDCl ) d 1.43 (s,

3

CH S), 2.05 (s, 3H, CH S), 2.32 (m, 1H, CH); 2.47–2.58

2

3

t

9

H, Bu), 1.86 (m, 1H, CHHS), 2.03 (m, 1H, CHHS), 2.10

(m, 3H, CH and CH ), 2.77 (m, 2H, CH ), 3.15–3.43 (m,

2 2

(

CHCO), 3.39 (m, 2H, CH N), 4.78 (s, 2H, OCH ), 4.89 (br

s, 1H, NH); C NMR (75 MHz, CDCl ) d 15.4, 28.5, 28.6,

3

s, 3H, SCH ), 2.60 (t, J¼7.5 Hz, 2H, CH ), 2.95 (m, 1H,

4H, 2£CH ), 4.21 (t, J¼6.6 Hz, 1H, CHCH O), 4.36 (d,

3

2

J¼6.5 Hz, 2H, CHCH O), 7.11 (m, 1H, NH), 7.28–7.40 (m,

2

1

3

4H, arom), 7.64 (d, J¼7.2 Hz, 2H, arom.), 7.79 (d,

1

3

1.6, 41.3, 44.7, 74.0, 79.7, 94.8, 155.7, 172.6; MALDI

þ

found: 416.0233.

J¼7.5 Hz, 2H, arom.), 8.05 (br s, 1H, NH); C NMR

HRMS calcd for C H Cl NO SNa (MþNa) : 416.0227;

(75 MHz, CD OD) d 15.0, 28.1, 29.9, 32.1, 36.0, 41.2, 43.6,

3

1

3

22

3

4

44.0, 45.3, 47.8, 67.5, 81.6, 120.5, 125.8, 127.7, 128.3,

142.1, 144.8, 144.9, 172.0, 175.0, 176.8; MALDI HRMS

2

t

2

þ

2

.3.5. Fmoc-(S)b hAsp(O Bu)-(S)b hMet-OCH CCl

2

calcd for C H N O SNa (MþNa) 593.2292, found

30 38 2 7

3

(

in CH Cl (6 mL), was added slowly TFA (6 mL) and the

16). To a solution of compound 15 (0.63 g, 1.60 mmol)

593.2299 (Fig. 24).

2

mixture stirred for 2 h at 25 8C. After removal of solvent

under reduced pressure, the amino ester was coupled with

2.3.7. Fmoc-(S)b hHis(Trt)-(S)b hLeu-OBn (17). Fmoc-

2

57

(S)b hHis(Trt)-OH (0.72 g, 1.34 mmol) and pTsOH·H-

2

t

56

65

Fmoc-(S)b hAsp(O Bu)-OH (0.68 g, 1.60 mmol) accord-

ing to GP1a. FC (AcOEt/hexane 1:1) yielded 16 (0.57 g,

(S)b hLeu-OBn

(0.49 g, 1.41 mmol) were coupled

according to GP1a in the presence of 4 equiv. of NMM.

The crude product was puriﬁed by FC (AcOEt/hexane 3:1)

5

1%) as a white solid. R ¼0.33 (AcOEt/hexane 1:1);

f

[

3

1

a] ¼þ12.8 (c¼0.49, CHCl ); IR (CHCl ) n

3445 (w),

008 (w), 2974 (w), 2923 (w), 1720 (s), 1667 (m), 1513 (m),

450 (m), 1368 (m), 1153 (s), 1077 (w), 1046 (w), 841

to yield 0.96 g (84%) of 17 as an amorphous solid. R ¼0.12

D

3

max

f

(AcOEt/hexane 3:1). [a] ¼22.5 (c¼0.75, CHCl ); IR

D

3

(CHCl ) n

3

3734 (w), 3628 (w), 3063 (w), 2923 (w),

max

2

1

t

(

w) cm ; H NMR (300 MHz, CDCl ) d 1.44 (s, 9H, Bu),

3

2853 (w), 1718 (s), 1652 (m), 1539 (m), 1495 (w), 1448 (s),

1 1

1

.80 (m, 1H, CHHS), 2.03 (m, 1H, CHHS), 2.08 (s, 3H,

CH S), 2.37 (m, 1H, CH), 2.54 (m, 3H, CH and CH ), 2.81–

1169 (m), 1245 (m), 1139 (m), 1085 (w), 843 (m) cm²; H

NMR (400 MHz, DMSO-d ) d 0.76 (d, J¼6.3 Hz, 3H, Me);

3

2

6

3

.05 (m, 2H, CH ), 3.29–3.57 (m, 4H, 2£CH N), 4.20 (t,

0.77 (d, J¼6.3 Hz, 3H, Me) 1.18–1.23 (m, 1H, CHH(i-Pr)),

2

J¼6.2 Hz, 1H, CHCH O), 4.39 (d, J¼6.2 Hz, 2H,

1.37–1.47 (m, 2H, CHH(i-Pr), Me CH); 2.52–2.62 (m, 4H,

2

CHCH O), 4.67–4.83 (m, 2H, CH CCl ), 5.46 (br s, 1H,

2

2£CHCO and CH Ar), 2.66–2.73 (m, 1H, CHHN); 2.97–

2

3

2

NH), 6.57 (br s, 1H, NH), 7.26–7.42 (m, 4H, arom.), 7.59

d, J¼7.1 Hz, 2H, arom.), 7.76 (d, J¼7.2 Hz, 2H, arom.);

3.06 (m, 1H, CHHN), 3.08–3.15 (m, 1H, CHHN), 3.20–

3.26 (m, 1H, CHHN), 4.16–4.25 (m, 3H, OCH CH), 5.00–

(

1

2

3

C NMR (75 MHz, CDCl ) d 15.4, 28.1, 28.7, 31.4, 35.7,

3

5.10 (m, 2H, CH Ph), 6.60 (s, 1H, arom.), 7.05–7.07 (m,

2

3

1

9.9, 42.2, 42.6, 44.1, 47.3, 66.7, 68.4, 74.1, 81.4, 119.9,

25.0, 126.9, 127.6, 141.2, 143.7, 156.4, 171.3, 172.2,

73.6; MALDI HRMS calcd for C H Cl N O SNa

6H, arom.), 7.21 (s, 1H, arom.), 7.27–7.41 (m, 19H, 18

arom., NH), 7.66–7.68 (m, 2H, arom.), 7.88–7.99 (m, 2H,

arom.), 8.02 (s, 1H, NH); C NMR (100 MHz, DMSO-d ) d

6

21.7, 22.7, 25.5, 28.6, 38.3, 40.6, 42.1, 43.2, 45.7, 46.6,

65.5, 74.3, 118.3, 120.0, 125.1, 125.1, 126.9, 127.5, 127.8,

1

3

2

39

3

2

7

þ

(

MþNa) : 723.1436; found: 723.1444.

2

t

2

2.3.6. Fmoc-(S)b hAsp(O Bu)-(S)b hMet-OH (5). Dipep-

tide ester 16 (0.86 g, 1.22 mmol) was dissolved in a mixture

of AcOH (50 mL) and H O (5 mL). To the cooled solution

2

127.8, 128.0, 128.3, 129.1, 136.0, 137.2, 138.4, 140.6,

142.2, 143.7, 156.0, 173.0, 173.9. MALDI HRMS calcd for

þ

C H N O Na (MþNa) : 873.3992; found: 873.3986.

55 54 4 5

2

Figure 25. Preparation of Fmoc-(S)b hHis(Trt)-(S)b hLeu-OH (6).

D. Seebach et al. / Tetrahedron 60 (2004) 7455–7506

7473

2

Figure 26. Preparation of Fmoc-(S,S)b hIle-(S)b hLys(Boc)-OH (7).

2

2.3.8. Fmoc-(S)b hHis(Trt)-(S)b hLeuOH (6). The C-

terminus of the fully protected dipeptide 17 (0.96 g,

(hexane/AcOEt 8:2!5:5) to yield 18 (1.13 g, 86%) as a

colorless solid; mp 93–96 8C; R ¼0.18 (AcOEt/hexane

f

1

8

9

.14 mmol) was deprotected according to GP2 in THF for

h. The crude product was puriﬁed by FC (CH Cl /MeOH

:1) to yield 0.78 g (90%) of 6 as an amorphous solid.

1:1); [a] ¼þ30.3 (c¼1.0, CHCl ); IR (CHCl ) n

3450

max

D

3

(w), 3007 (w), 2968 (m), 2872 (w), 1713 (s), 1666 (m), 1509

(s), 1456 (m), 1367 (m), 1170 (m), 1064 (w) cm²; H NMR

2

1 1

R ¼0.18 (CH Cl /MeOH 9:1); [a] ¼þ10.6 (c¼0.43,

(500 MHz, CDCl ) d 0.87 (t, J¼7.4 Hz, 3H, Me), 0.91 (d,

f

2

D

3

CHCl ); IR (CHCl ) n 3297 (w), 3061 (w), 2922 (w),

max

J¼6.9 Hz, 3H, Me), 1.08–1.19 (m, 1H, CH), 1.30–1.37 (m,

3

t

2

1

854 (w), 1719 (s), 1649 (s), 1551 (s), 1449 (s), 1245 (m),

1

2H, CH ), 1.43 (s, 9H, Bu), 1.42–1.51 (m, 2H, CH ), 1.52–

2

132 (m), 1001 (w), 842 (m) cm²; H NMR (400 MHz,

1

1.58 (m, 2H, CH ),1.59–1.67 (m, 2H, CH ), 2.22–2.26 (m,

2 2

DMSO-d ) d 0.77 (d, J¼6.4 Hz, 3H, Me); 0.79 (d,

1H, CH), 2.60–2.66 (m, 1H, CH), 3.07 (d, J¼6.0 Hz, 2H,

6

J¼6.4 Hz, 3H, Me) 1.12–1.14 (m, 1H, CHH(i-Pr)); 1.36–

CH ), 3.26–3.35 (m, 2H, CH ), 3.42–3.54 (m, 2H, CH ),

2

1

.44 (m, 1H, CHH(i-Pr)), 1.52–1.53 (m, 1H, Me CH),

2

3.68 (s, 3H, MeO), 4.55 (br s, 1H, NH), 5.07 (q, J¼12.4 Hz,

2

.40–2.46 (m, 1H, CHCO), 2.52–2.69 (m, 3H, CH Ar,

2

2H, CH Ph), 5.35 (br s, 1H, NH), 6.10 (br s, 1H, NH), 7.30–

2

1

3

CHCO), 2.96–3.17 (m, 4H, 2£CH N), 4.16–4.25 (m, 3H,

7.36 (m, 5H, arom.); C NMR (125 MHz, CDCl ) d 11.4,

3

2

OCH CH), 6.64 (s, 1H, arom.), 7.05–7.07 (m, 6H, arom.),

2

15.9, 23.7, 27.2, 28.4, 28.9, 29.4, 29.7, 29.9, 35.2, 39.9,

40.2, 40.4, 44.8, 45.1, 51.9, 52.0, 58.5, 66.6, 72.3, 79.2,

128.0, 128.5, 136.6, 156.1, 156.5, 174.4, 175.4; MALDI

7

.21 (s, 1H, arom.), 7.28–7.44 (m, 14H, 13 arom., NH),

.67–7.68 (m, 2H, arom.), 7.86–7.88 (m, 2H, arom.), 7.97

1

3

þ

(s, NH), 12.35 (br s, 1H, OH); C NMR (100 MHz, DMSO-

d₆) d 21.8, 22.8, 25.5, 28.4, 38.5, 40.9, 41.8, 45.4, 46.6, 54.8,

HRMS calcd for C H N O Na (MþNa) : 558.3150,

2

8 45 3 7

found: 558.3157. Anal. calcd for C H N O : C 62.78, H

28 45 3 7

8.47, N 7.84; found: C 62.80, H 8.28, N 7.71.

6

1

7

5.3, 74.4, 119.9, 121.3, 127.0, 127.2, 127.5, 127.8, 128.0,

28.8, 129.2, 138.1, 140.6, 140.6, 142.2, 143.8, 156.0,

þ

2

72.8; MALDI HRMS calcd for C H N O Na (MþNa) :

2.3.10. Fmoc-(S,S)b hIle-(S)b hLys(Boc)-OH (7). The

methyl ester- and the Cbz-protecting group of compound 18

4

8 48 4 5

83.3523; found: 783.3517 (Fig. 25).

(

1.06 g, 1.98 mmol) were removed according to GP3 and

2

.3.9. Cbz-(S,S)b hIle-(S)b hLys(Boc)-OMe (18). The

2

GP2 respectively, and the resulting amino acid Fmoc-

protected according to GP4. FC (CH Cl /MeOH

54

Cbz-protecting group of Cbz-(S)b hLys(Boc)-OMe

1.01 g, 2.47 mmol) was hydrogenated according to GP2

2

(

and the resulting crude H-(S)b hLys(Boc)-OMe was

100:1!CH Cl /MeOH/AcOH 20:1:0.2) yielded 7 (0.94 g,

2

78%) colorless solid; mp 114–117 8C; R ¼0.30 (CH Cl /

D 3

f 2 2

2

55

coupled with Cbz-(S,S)b hIle-OH (0.69 g, 2.47 mmol)

according to GP1b. The crude product was puriﬁed by FC

MeOH/AcOH 20:1:0.2); [a] ¼þ29.4 (c¼1.0, CHCl ); IR

(CHCl ) n 3449 (w), 3344 (w), 3007 (w), 2969 (m), 2933

3 max

2

Figure 27. Preparation of Boc-(S)b hCys(Trt)-(S)b hGln(Trt)-OH (8).

7474

D. Seebach et al. / Tetrahedron 60 (2004) 7455–7506

t

(

m), 2872 (w), 1710 (s), 1662 (m), 1510 (s), 1451 (m), 1368

1

CH Si), 1.31 (s, 9H, Bu), 1.61–1.64 (m, 2H, CH CH

2 2 2-

2

1

m), 1167 (m), 1078 (w), 990 (w) cm

;

H NMR

CON), 2.01–2.03 (m, 1H,CHHS), 2.25–2.28 (m, 2H,

CH CON), 2.30–2.33 (m, 2H, CHHS and CHCO), 2.47–

2.50 (m, 1H, CHCO), 2.86–2.90 (m, 2H, CH N), 3.11–3.25

2

500 MHz, CD OD) d 0.89 (t, J¼7.4 Hz, 3H, Me), 0.94 (t,

3

2

J¼6.8 Hz, 3H, Me), 1.10–1.57 (m, 2H, CH ), 1.29–1.39

2

t

(

m, 2H, CH ), 1.41 (s, 9H, Bu), 1.42–1.49 (m, 2H, CH ),

2

(m, 2H, CH N), 4.00–4.14 (m, 2H, OCH ), 6.49 (s, 1H,

2

1

2

.51–1.63 (m, 4H, 2£CH ), 2.29–2.34 (m, 1H, CH), 2.58–

NH), 7.15–7.30 (m, 30H, arom.), 7.93 (s, 1H, NH), 8.54 (s,

1

2

3

.62 (m, 1H, CH), 3.00 (d, J¼6.9 Hz, 2H, CH ), 3.20–3.28

1H, NH); C NMR (100 MHz, DMSO-d ) d 21.6, 16.8,

6

2

(

2

1

m, 2H, CH ), 3.32–3.39 (m, 2H, CH ), 4.19 (t, J¼6.9 Hz,

25.1, 28.1, 31.1, 33.1, 39.9, 42.0, 44.3, 45.4; 61.9, 65.8,

69.1, 77.5, 126.2, 126.5, 127.3, 127.8, 128.4, 129.0, 144.3,

144.8, 155.2, 171.1, 171.6, 173.5; MALDI HRMS calcd for

2

H, CH ), 4.31 (d, J¼7.1 Hz, 2H, CH ), 6.89 (t, J¼5.9 Hz,

2

1

3

H, NH), 7.29–7.80 (m, 8H, arom.); C NMR (125 MHz,

þ

CD OD) d 11.7, 16.5, 25.3, 28.3, 28.8, 30.7, 30.9, 36.5,

3

C H N O SSiNa (MþNa) : 984.4418; found: 984.4400.

58 67 3 6

4

1

1.1, 41.8, 42.0, 46.6, 48.5, 53.4, 67.9, 73.6, 79.8, 121.0,

26.2, 126.3, 128.2, 128.8, 142.6, 145.3, 145.4, 158.6,

58.7, 176.9, 178.2; MALDI HRMS calcd for

2

2.3.13. Boc-(S)b hCys(Trt)-(S)b hGln(Trt)-OH (8). The

C-terminus of the fully protected dipeptide 20 (1.32 g,

1.37 mmol) was deprotected according to GP2. The crude

product was puriﬁed by FC (AcOEt/Hexane/AcOH

20:10:0.1) to yield 8 (1.00 g, 85%) as an amorphous solid.

R ¼0.10 (AcOEt/hexane/AcOH 20:10:0.1); [a] ¼26.3

þ

C H N O Na (MþNa) : 632.3306; found: 632.3313.

3

4 47 3 7

Anal. calcd for C H N O : C 66.97, H 7.77, N 6.89;

3

4 47 3 7

found: C 66.97, H 7.66, N 6.64 (Fig. 26).

f

D

2

.3.11.

2

Cbz-(S)b hGln(Trt)-OTMSE

56

(19).

S)b hGln(Trt)-OBn (2.45 g, 3.9 mmol) was transformed

Cbz-

(c¼0.9, CHCl ); IR (CHCl ) n

3432 (w), 3058 (w),

max

3

(

to compound 19 in three steps. The ﬁrst transformation

3009 (w), 2980 (w), 1706 (s), 1667 (m), 1494 (s), 1445 (m),

2

1 1

1366 (w), 1228 (m), 1164 (m) cm ; H NMR (400 MHz,

t

involved hydrogenolysis in MeOH for 1.5 h according to

2

GP2 to yield the amino acid (H-b hGln(Trt)-OH), which

DMSO-d ) d 1.31 (s, 9H, Bu), 1.62–1.66 (m, 2H,

6

CH CH CON), 2.02–2.05 (m, 1H, CHHS), 2.28–2.33 (m,

2

without puriﬁcation was N-Cbz protected using the follow-

ing procedure: to an aqueous solution of the free amino acid

in 0.5 M NaOH (1.1 equiv.) at 0 8C was added with

4H, CHHS, CH CON, and CHCO), 2.37–2.43 (m, 1H,

2

CHCO), 2.84–2.88 (m, 2H, CH N), 3.06–3.10 (m, 1H,

2

CHHN), 3.25–3.31 (m, 1H, CHHN), 6.50 (t, J¼5.8 Hz, 1H,

continuous stirring BnO CCl (1.3 equiv.). The pH was

2

NH), 7.15–7.32 (m, 30H, arom.), 7.89 (t, J¼5.8 Hz, 1H,

13

kept basic by periodic addition of 1 M NaOH. After the

addition was completed, the reaction mixture was stirred for

one additional hour then extracted with AcOEt after

adjusting the pH to 3 using 1 M HCl. The organic layer

NH), 8.57 (s, 1H, NH), 12.30 (br s, 1H, OH); C NMR

(100 MHz, DMSO-d ) d 25.1, 28.1, 31.1, 33.4, 41.3, 42.1,

6

44.3, 45.4, 65.8, 69.1, 77.5, 126.2, 126.5, 127.3, 127.8,

128.4, 129.0, 144.3, 144.8, 155.3, 171.3, 171.6, 175.2;

þ

was washed with brine, dried (MgSO ) and the solvent

4

MALDI HRMS calcd for C H N O SNa (MþNa) :

53 55 3 6

evaporated under reduced pressure to give a yellow oil.

Unreacted BnO CCl and BnOH were removed from the

884.3708; found: 884.3704 (Fig. 27).

2

t

2

mixture by passing the mixture through a short column, and

the crude product treated according to GP5. The puriﬁcation

by FC (Et O/pentane 1:3) yielded 19 (1.52 g, 61%) as a

2.3.14. Cbz-(S)b hTyr( Bu)-(S)b hAla-OBn (21). H-

2

89

(S)b hAla-OH was dissolved in toluene, BnOH and

pTsOH were added and the mixture was heated at reﬂux

for 18 h using a Dean–Stark trap to azeotropically remove

2

white solid; mp 135–136 8C; R ¼0.42 (Et O/pentane 1:3);

f

2

[

a] ¼26.8 (c¼1.0, CHCl ); IR (CHCl ) n

3439 (w),

007 (w), 2956 (w), 1716 (s), 1516 (m), 1490 (m), 1448 (w),

H O. The reaction mixture was cooled to 25 8C, toluene was

2

D

3

max

3

8

0

evaporated and the residue washed several times with

Et O. The resulting benzyl ester (0.92 g, 2.52 mmol) was

2

1 1

60 (w), 839(w) cm ; H NMR (400 MHz, DMSO-d ) d

6

2

t

55

.01 (s, 9H, SiMe ), 0.90–0.95 (m, 2H, CH Si); 1.56–1.65

3

coupled

with

Cbz-(S)b hTyr( Bu)-OH

(0.97 g,

2

(

m, 2H, CH CH CON), 2.24–2.27 (m, 2H, CH CON),

2

.44–2.47 (m, 1H, CHCO), 3.06–3.19 (m, 2H, CH N),

2

2.52 mmol) according to GP1a. FC (AcOEt/hexane 1:1)

yielded 21 (1.17 g, 83%) as a white solid; mp 74–76 8C;

R ¼0.18 (AcOEt/hexane 1:1); [a] ¼þ14.8 (c¼0.54,

2

4

7

.06–4.11 (m, 2H, OCH ), 4.95–5.02 (m, 2H, CH Ph),

2

f

D

1

3

.14–7.35 (m, 21H, 20 arom., NH), 8.53 (s, 1H, NH);

C

CHCl ); IR (CHCl ) n 3443 (w), 2980 (m), 1719 (s),

max

3

NMR (100 MHz, DMSO-d ) d21.5, 16.8, 25.0, 33.2, 42.0,

6

1667 (m), 1507 (s), 1456 (m), 1367 (m), 1160 (m), 894

1

(m) cm²

;

1

H NMR (400 MHz, CD OD) d 0.96 (d,

3

4

1

4.9, 62.0, 65.2, 69.2, 126.2, 127.4, 127.6, 127.7, 128.3,

28.5, 137.1, 144.9, 156.1, 171.2, 173.5; MALDI HRMS

t

J¼7.1 Hz, 3H, Me), 1.28 (s, 9H, Bu), 2.54 (q, J¼7.0 Hz,

þ

calcd for C H N O SiNa (MþNa) : 659.2917; found:

1H, CHCO), 2.66–2.93 (m, 3H, CHCH Ph), 3.11–3.29 (m,

2

3

8

44

2

5

6

59.2912.

4H, 2£CH N), 5.05 (s, 2H, OCH Ph), 5.07 (AB, J¼12.6 Hz,

2

H, OCH Ph), 6.85 (d, J¼8.4 Hz, 2H, arom.), 7.04 (d,

2

13

2

.3.12. Boc-(S)b hCys(Trt)-(S)b hGln(Trt)-OTMSE

20). Compound 19 (1.00 g, 1.57 mmol) was hydrogenated

J¼8.4 Hz, 2H, arom.), 7.26–7.34 (m, 10H, arom.); C

NMR (100 MHz, CD OD) d 15.2, 29.2, 36.5, 40.8, 42.9,

(

in THF to give H-(S)b hGln(Trt)-OTMSE according to

GP2, then coupled with Boc-(S)b hCys(Trt)-OH (0.75 g,

.57 mmol) according to GP1a. The crude product was

puriﬁed by FC (CH Cl /i-PrOH 96:4) to yield 20 (1.22 g,

3

2

44.1, 50.4, 67.4, 67.5, 79.4, 125.2, 128.8, 129.0, 129.1,

129.2, 129.5, 129.6, 130.4, 135.5, 137.6, 138.3, 155.0,

158.8, 176.0; MALDI HRMS calcd for C H N O Na

2

57

1

3

3 40 2 6

þ

(MþNa) 583.2779, found 583.2785. Anal. calcd for

2

8

9

1%) as an amorphous solid; R ¼0.32 (CH Cl /i-PrOH

C H N O : C 70.69, H 7.19, N 5.00; found: C 70.52, H

33 40 2 6

7.34, N 5.14.

f

2

6:4); [a] ¼þ11.4 (c¼1.0, CHCl ); IR (CHCl ) n

3436

max

D

3

(

w), 3026 (w), 1708 (s), 1681 (m), 1492 (s), 1446 (w), 1251

m), 1164 (m), 1041 (w) cm

2

1

2

t

2

;

H NMR (400 MHz,

2.3.15. Fmoc-(S)b hTyr( Bu)-(S)b hAla-OH (9). The

dipeptide derivative 21 was hydrogenated according to

DMSO-d ) d 0.00 (s, 9H, SiMe ) 0.93 (t, J¼8.5 Hz, 2H,

6

3

D. Seebach et al. / Tetrahedron 60 (2004) 7455–7506

7475

2

Figure 28. Preparation of Fmoc-(S)b hTyr( Bu)-(S)b hAla-OH (8).

t

2

1

3

GP2 and Fmoc-protected according to GP4. The crude

peptide was puriﬁed by FC (AcOEt/hexane/AcOH

J¼4.6 Hz, 1H, NH); C NMR (100 MHz, DMSO-d ) d

6

20.1, 27.0, 28.2, 37.3, 39.7, 46.1, 52.6, 60.3, 65.0, 65.2,

66.2, 72.0, 73.2, 127.4, 127.6, 127.6, 127.8, 128.2, 128.2,

136.1, 137.2, 155.9, 171.7, 172.2; MALDI HRMS calcd for

C H N O Na: 593.3203; found: 593.3197.

1

0:10:0.1) to give 9 (0.72 g, 73%) as a white solid; mp

43–144 8C; R ¼0.16 (AcOEt/hexane/AcOH 10:10:1);

f

[

a] ¼þ7.3 (c¼0.55, CHCl ); IR (CHCl ) n

3442 (w),

005 (m), 2980 (m), 1717 (s), 1683 (m), 1508 (s), 1467 (m),

450 (m), 1368 (m), 1157 (m), 1082 (w), 891 (m) cm²¹; ¹H

D

3

max

32 46 2 7

3

1

2

t

2

t

2.3.17. Fmoc-(R,S)b hThr( Bu)-(R)b hSer( Bu)-OH

(10). The fully protected dipeptide 22 (0.9 g, 1.58 mmol)

was hydrogenolyzed in MeOH for 16 h according to GP2.

The resulting amino acid was Fmoc-protected according to

GP4. The crude product was puriﬁed by FC (AcOEt/

Hexane/AcOH 10:10:0.1) to obtain 0.73 g (81%) of 22 as

NMR (400 MHz, CD OD) d 0.98 (d, J¼7.1 Hz, 3H, Me),

3

t

1

.29 (s, 9H, Bu), 2.45 (q, J¼7.1 Hz, 1H, CHMe), 2.67–2.80

(

m, 3H, CHHN, CH Ph), 3.15 (dd, J¼7.0, 13.4 Hz, 1H,

2

CH), 3.26 (m, 3H, CH N, CHHN), 4.19 (t, J¼6.8 Hz, 1H,

2

CHCH O), 4.34 (d, J¼6.9 Hz, 2H, CHCH O), 6.87 (d,

2

J¼8.5 Hz, 2H, arom.), 7.07 (d, J¼8.4 Hz, 2H, arom.), 7.29

colorless oil. R ¼0.20 (AcOEt/hexane/AcOH 10:10:0.1).

f

(

arom.), 7.77 (m, 2H, arom.); C NMR (100 MHz, CD OD)

m, 2H, arom.), 7.37 (m, 2H, arom.), 7.63 (d, J¼7.5 Hz, 2H,

[a] ¼þ12.7 (c¼0.52, CHCl ); IR (CHCl ) n

3312 (w),

max

D

3

1

3

2971 (m), 2925 (w), 1718 (s), 1653 (m), 1539 (m), 1450 (m),

3

1363 (m), 1249 (m), 1193 (m), 1091 (w) cm²; H NMR

1 1

d 15.4, 29.2, 36.5, 40.6, 43.0, 44.2, 48.5, 50.4, 67.8, 79.5,

1

21.0, 125.3, 126.2, 128.2, 128.8, 130.5, 135.7, 142.7,

45.4, 155.0, 158.9, 176.1, 178.4; MALDI HRMS calcd for

(400 MHz, DMSO-d ) d 1.00 (d, J¼4.8 Hz, 3H, Me), 1.08

6

t

(s, 9H, Bu), 1.12 (s, 9H, Bu), 2.36–2.43 (m, 1H, CHCO),

2.53–2.60 (m, 1H, CHCO), 3.12–3.22 (m, 3H, CH N,

þ

C H N O Na (MþNa) : 581.2622, found: 581.2629.

3

38

2

6

2

Anal. calcd for C H N O : C 70.95, H 6.86, N 5.01;

3

OCHH), 3.35–3.41 (m, 1H, OCHH), 3.45 (d, J¼4.6 Hz, 2H,

3 38 2 6

found: C 70.99, H 6.87, N 4.89 (Fig. 28).

CH N); 3.69–3.74 (m, 1H, OCH), 4.18–4.27 (m, 3H,

2

OCH CH), 7.12 (t, J¼4.5 Hz, 1H, NH), 7.30–7.34 (m, 2H,

2

t

2

t

2

Cbz-(R,S)b hThr( Bu)-OH (0.70 g, 2.16 mmol) and H-

(

.3.16. Cbz-(R,S)b hThr( Bu)-(R)b hSer( Bu)-OBn (22).

2

arom.), 7.39–7.43 (m, 2H, arom.), 7.62 (t, J¼4.5 Hz, 1H,

NH), 7.67 (d, J¼5.8 Hz, 2H, arom.), 7.88 (d, J¼6.1 Hz, 2H,

t

57

2

t

57

13

R)b hSer( Bu)-OBn (0.6 g, 2.27 mmol) were coupled

according to GP1a. The crude product was puriﬁed by FC

AcOEt/hexane 3:4) to obtain 0.97 g (79%) of 22 as an

arom.), 12.22 (br s, 1H, OH); C NMR (100 MHz, DMSO-

d₆) d 20.1, 27.1, 28.1, 37.4, 39.9, 46.0, 46.6, 52.4, 60.4, 65.4,

66.3, 72.2, 73.3, 120.0, 125.1, 126.9, 127.5, 140.6, 143.8,

155.9, 171.6, 173.8; MALDI HRMS calcd for

C H N O Na: 591.3047; found: 591.3041 (Fig. 29).

(

amorphous solid. R ¼0.35 (AcOEt/hexane 3:4);

f

[

a] ¼þ27.6 (c¼0.41, CHCl ); IR (CHCl ) n

3323 (w),

972 (m), 2925 (w), 1726 (s), 1653 (m), 1534 (m), 1457 (m),

D

3

max

32 44 2 7

2

1

233 (m), 1193 (m), 1091 (w) cm²; H NMR (400 MHz,

1

2.3.18. Cbz-(S)b hGlu(O Bu)-OTMSE (23). Cbz-

2

t

56

DMSO-d ) d 0.97 (d, J¼4.8 Hz, 3H, Me); 1.05 (s, 9H, Bu),

(S)b hGlu(O Bu)-OH (0.9 g, 2.56 mmol) was treated

according to GP5. Puriﬁcation of the crude product by FC

6

t

1

.10 (s, 9H, Bu), 2.36–2.40 (m, 1H, CHCO), 2.74–2.79 (m,

H, CHCO), 3.10–3.27 (m, 3H, CH N, OCHH), 3.33–3.37

(Et O/pentane 1:1) gave 23 (1.06 g, 92%) as a colorless oil.

2

(

1

m, 1H, OCHH), 3.44–3.51 (m, 2H, CH N), 3.68–3.70 (m,

2

R ¼0.43 (Et O/pentane 1:1). [a] ¼þ4.7 (c¼0.9, CHCl );

f

2

D

3

H, OCH), 4.95–5.17 (m, 4H, 2£CH Ph), 7.03 (t,

IR (CHCl ) n

3

3446 (w), 3025 (w), 2954 (w), 1718 (s),

max

2

J¼4.6 Hz, 1H, NH) 7.27–7.37 (m, 10H, arom.); 7.72 (t,

1508 (m), 1451 (w), 1364 (w), 1246 (m), 1148 (m), 1056

2

t

2

Figure 29. Preparation of Fmoc-(R,S)b hThr( Bu)-(R)b hSer( Bu)-OH (10).

t

7476

D. Seebach et al. / Tetrahedron 60 (2004) 7455–7506

2

Figure 30. Preparation of Fmoc-(S)b hAsn(Trt)-(S)b hGlu(O Bu)-OH (11).

2

t

(

w), 856 (w) cm²¹; ¹H NMR (400 MHz, DMSO-d ) d 0.02

arom., NH), 7.66 (d, J¼7.5 Hz, 2H, arom.), 7.87 (d,

J¼7.6 Hz, 2H, arom.), 7.89 (s, 1H, NH), 8.52 (s, 1H, NH);

C NMR (100 MHz, DMSO-d ) d 21.6, 16.7, 24.1, 27.6,

6

t

s, 9H, SiMe ), 0.91–0.95 (m, 2H, CH Si), 1.39 (s, 9H, Bu),

2

3

1

3

1

CH CO), 2.24–2.27 (m, 2H, CH CON), 2.51–2.55 (m,

1

OCH ), 5.00 (s, 2H, CH Ph), 7.28–7.40 (m, 6H, 5 arom.,

NH); C NMR (100 MHz, DMSO-d ) d 21.6, 16.7,

6

2

1

.62–1.73 (m, 2H, CH CH CO), 2.11–2.26 (m, 2H,

2

32.0, 33.1, 36.0, 40.1, 42.3, 42.4, 43.9, 46.6, 62.0, 65.4,

69.2, 79.5, 119.9, 121.3, 125.1, 126.9, 127.2, 127.3, 128.4,

140.6, 143.8, 144.8, 155.9, 170.1, 171.5, 172.7, 173.4;

2

H, CHCO), 3.07–3.23 (m, 2H, CH N), 4.07–4.11 (m, 2H,

2

1

3

þ

MALDI HRMS calcd for C H N O SiNa (MþNa) :

54 63 3 8

4.2, 27.6, 32.1, 41.7, 44.4, 62.0, 65.1, 79.6, 127.5,

27.7, 128.2, 137.0, 156.0, 171.4, 173.2; MALDI HRMS

932.4282; found: 932.4277.

þ

2

t

calcd for C H NO SiNa (MþNa) : 474.2388; found:

2.3.20. Fmoc-(S)b hAsn(Trt)-(S)b hGlu(O Bu)-OH (11).

The C-terminus of dipeptide derivative 24 (1.15 g,

2

3

37

6

4

74.2382.

1

.27 mmol) was deprotected according to GP6; however,

2

t

2

.3.19. Fmoc-(S)b hAsn(Trt)-(S)b hGlu(O Bu)-OTMSE

24). Compound 23 (0.93 g, 2.06 mmol) was hydrogenated

considerable amounts of Fmoc-deprotection took place

during the reaction. For this reason, the crude reaction

mixture was evaporated under reduced pressure and treated

in situ with FmocOSu (1.2 equiv.) according to GP4. The

crude product was puriﬁed by FC (AcOEt/CH Cl 1:7!1:3)

(

according to GP2 in THF. Coupling with Fmoc-

2

56

(

S)b hAsn(Trt)-OH (1.26 g, 2.06 mmol) according to

GP1a give a crude product, which was puriﬁed by FC

AcOEt/hexane 1:1) to yield 24 (1.39 g, 74%) as an

2

(

to give 11 (0.74 g, 72%) as an amorphous solid. R ¼0.23

f

amorphous solid. R ¼0.23 (AcOEt/hexane 1:1).

(AcOEt/hexane 1:1); [a] ¼211.2 (c¼0.75, CHCl ); IR

f

D

3

[

(

a] ¼216.0 (c¼0.43, CHCl ); IR (CHCl ) n

3436

w), 3019 (w), 1716 (s), 1677 (m), 1504 (m), 1445 (w), 1248

(CHCl ) n

3

3426 (w), 3343 (w), 3015 (w), 2985 (w), 1718

max

D

3

max

(s), 1677 (m), 1508 (m), 1492 (m), 1446 (w), 1369 (w), 1246

21 1

2

1

w), 1154 (m) cm ; H NMR (400 MHz, DMSO-d ) d

6

(m), 1154 (m) cm ; H NMR (400 MHz, DMSO-d ) d 1.35

6

t

2

0.01 (s, 9H, SiMe ), 0.89–0.94 (m, 2H, CH Si), 1.34 (s,

3

H, Bu), 1.64–171 (m, 2H, CH CH CO), 2.07–2.21 (m,

2

H, CH CO), 2.27–2.33 (m, 1H, CHCO), 2.52–2.59 (m,

2

H, CHHCON and CHCO), 2.71–2.75 (m, 1H, CHHCON),

.99–3.05 (m, 1H, CHHN), 3.06–3.08 (m, 2H, CH N),

(s, 9H, Bu), 1.62–1.74 (m, 2H, CH CH CO), 2.11–2.25

2

(m, 2H, CH CO), 2.28–2.32 (m, 1H, CHCO), 2.43–2.48

2

(m, 1H, CHCO), 2.55–2.60 (m, 1H, CHHCON), 2.72–2.78

(m, 1H, CHHCON), 2.99–3.06 (m, 1H, CHHN), 3.07–3.14

(m, 2H, CH N), 3.35–3.41 (m, 1H, CHHN), 4.19–4.29 (m,

2

t

9

2

3

4

2

.33–3.39 (m, 1H, CHHN), 4.03–4.13 (m, 2H, OCH₂),

.18–4.29 (m, 3H, OCH CH), 7.13–7.41 (m, 20H, 19

3H, OCH CH), 7.13–7.42 (m, 21H, 19 arom., 2£NH), 7.68

2

(d, J¼6.0 Hz, 2H, arom.), 7.88 (d, J¼5.8 Hz, 2H, arom.),

2

Figure 31. Preparation of Fmoc-(S)b hArg(Boc) -(S)b hVal-OH (8).

D. Seebach et al. / Tetrahedron 60 (2004) 7455–7506

7477

1

3

8

(

4

1

.53 (s, 1H, NH), 12.30 (br s, 1H, OH); C NMR

100 MHz, DMSO-d ) d 24.2, 27.6, 32.2, 36.1, 40.0, 42.4,

173.1, 173.2; MALDI HRMS calcd for C H N O Na

3

8 53 5 9

þ

C H N O : C 63.05, H 7.38, N 9.67; found: C 63.17, H

7.40, N 9.44 (Fig. 31).

(MþNa) : 746.3736, found: 746.3728. Anal. calcd for

6

2.4, 43.8, 46.6, 65.4, 69.2, 79.5, 120.0, 125.1, 126.2, 127.0,

27.3, 127.5, 128.4, 140.6, 143.8, 144.8, 156.0, 170.1,

71.6, 172.7, 175.0; MALDI HRMS calcd for

C H N O Na (MþNa) : 832.3598; found: 832.3568

3

8 53 5 9

þ

2.4. Solid-phase synthesis of 2 and 3 and their ligation to

eicosapeptide 1

4

9 51 3 8

(

Fig. 30).

2

(

.3.21. Cbz-(S)b hArg(Boc) -(S)b hVal-OBn (25). H-

2

2.4.1. H-b hGly-(S)b hTrp-(S)b hArg-(S)b hVal-

2

89

2

S)b hVal-OH was dissolved in toluene, BnOH and

pTsOH were added and mixture was reﬂuxed for 18 h

using a Dean–Stark trap to azeotropically remove H O. The

2

reaction mixture was cooled to 25 8C, and the toluene

removed under reduced pressure. The crude product was

puriﬁed by recrystallization from toluene/hexane to give the

(S)b hAsn-(S)b hGlu-(R,S)b hThr-(R)b hSer-

2

(S)b hTyr-(S)b hAla-S-Ethylpropionate (2). The loading

of the sulfamylbutyryl resin was performed according to a

9

0

procedure found in the literature. A solution of Fmoc-

2

t

2

(S)b hTyr( Bu)-(S)b hAla-OH (9) (502 mg, 0.9 mmol),

DIPCDI (0.5 mL, 3.6 mmol) and 1-MeIm (0.2 mL,

3.6 mmol) in CH Cl /DMF (4:1) was added to the resin

2

pTsOH-salt of H-(S)b hVal-OBn (0.72 g, 1.82 mmol)

2

48

which was coupled with Cbz-(S)b hArg(Boc) -OH

2

(205 mg, 0.225 mmol) that had been preswelled in CH Cl

2 2

(

3

0.95 g, 1.82 mmol) according to GP1b. FC (AcOEt/hexane

:7) yielded 25 (1.06 g, 80%) as white solid; mp 45–47 8C;

for 1 h. The suspension was gently stirred using Ar bubbling

for 18 h at 25 8C. Consequently, the resin was ﬁltered off,

washed with DMF (4 mL, 4£1 min), CH Cl (4 mL,

R ¼0.22 (AcOEt/hexane 1:1); [a] ¼þ16.8 (c¼0.37,

f

D

2

CHCl ); IR (CHCl ) n 3446 (w), 3328 (w), 3007 (m),

max

4£1 min), and dried under h.v. overnight. Resin loading

3

2

1

(

974 (m), 2933 (w), 1719 (s), 1636 (s), 1615 (s), 1574 (m),

509 (m), 1418 (m), 1369 (m), 1332 (m), 1282 (m), 1136

was measured using the absorbance of the benzofulvene–

8

0

piperidine adduct according to Schreiber and Seebach and

was determined to be 0.66 mmol/g, which corresponds to

0.133 mmol of 9. The Fmoc group was removed using 20%

piperidine in DMF (4 mL, 4£10 min) under Ar bubbling.

After ﬁltration, the resin was washed with DMF (4mL,

4£1 min). Solid phase synthesis was continued by sequen-

tial incorporation of N-Fmoc-protected dipeptides or N-

2

1 1

s), 1051 (m), 1027 (m), 872(w), 648(w) cm ; H NMR

400 MHz, CDCl ) d 0.90 (d, J¼6.8 Hz, 3H, Me), 0.94 (d,

3

t

J¼6.8 Hz, 3H, Me), 1.41 (m, 2H, CH ), 1.47 (s, 9H, Bu),

2

t

1

.49 (s, 9H, Bu), 1.55 (m, 1H, CHH), 1.78 (m, 1H, CHH),

.93 (m, 1H, CH(Me) ), 2.47 (m, 1H, CHCO), 2.50 (m, 1H,

2

CHCO), 3.21–3.38 (m, 5H, 2£CH and CHH), 3.60 (m, 1H,

2

CHH), 5.04 (d, J¼12.2 Hz, 1H, OCHHPh), 5.08 (s, 2H,

Fmoc protected b -homoamino acids building blocks (10,

2

OCH Ph), 5.15 (d, J¼12.2 Hz, 1H, OCHHPh), 5.32 (br s,

11, 12, Fmoc-(S)b hTrp(Boc)-OH, Boc-b hGly-OH). For

each coupling step, the resin was treated with a solution of

Fmoc-protected building block (3 equiv.), HATU

(2.9 equiv.) and DIPEA (6 equiv.) in DMF. The suspension

was then gently stirred using Ar bubbling for 45–60 min.

Monitoring of the coupling reaction was performed with

2

1

8

(

3

1

H, NH), 6.30 (br s, 1H, NH), 7.28–7.52 (m, 10H, arom.),

.32 (br s, 1H, NH), 11.48 (br s, 1H, NH); C NMR

1

3

100 MHz, CDCl ) d 20.0, 20.3, 26.6, 27.1, 28.1, 28.3, 28.9,

3

1.3, 38.8, 40.1, 42.8, 45.8, 51.9, 66.3, 66.6, 79.3, 83.2,

28.0, 128.1, 128.4, 128.5, 128.6, 135.8, 136.6, 153.3,

56.3, 156.6, 163.4, 174.2, 174.4; MALDI HRMS calcd for

9

1

TNBS. In the case of a positive TNBS test (indicating

incomplete coupling), the suspension was ﬁltrated, and

treated again with a freshly prepared solution of the same

N-Fmoc protected building block (2 equiv.) and coupling

reagents. The resin was then ﬁltered off and washed with

DMF (4 mL, 4£1 min) prior to the subsequent Fmoc

deprotection step using 20% piperidine in DMF (4 mL,

4£10 min). After ﬁltration, the resin was washed with DMF

(4 mL, 3£1 min) and solid-phase synthesis was continued

by sequential incorporation of N-Fmoc protected building

blocks. For each coupling step, the resin was treated as

described above. After the last coupling the resin was

ﬁltered off, washed with DMF (4 mL, 4£1 min), CH Cl

þ

C H N O Na (MþNa) : 748.3892; found: 748.3901.

3

8 55 5 9

Anal. calcd for C H N O : C 62.88, H 7.64, N 9.65;

8 55 5 9

3

found: C 62.92, H 7.68, N 9.51.

2

.3.22. Fmoc-(S)b hArg(Boc) -(S)b hVal-OH (12).

2

Dipeptide derivative 25 (1.06 g) was hydrogenated accord-

ing to GP2. Subsequent Fmoc-protection according to GP4

and FC (AcOEt/hexane 1:1!1:0) yielded 12 (0.99 g, 78%)

as white solid; mp 93–96 8C; R ¼0.46 (AcOEt/hexane/

f

AcOH 10:10:1); [a] ¼þ29.3 (c¼0.91, CHCl ); IR (CHCl )

D

3

n

1

6

3446 (w), 3326 (w), 3007 (m), 2972 (m), 2934 (m),

719 (s), 1643 (s), 1615 (s), 1513 (m), 1450 (m), 1416 (m),

369 (m), 1333 (m), 1282 (m), 1138 (s), 1053 (m), 872 (w),

max

2

(4 mL, 4£1 min), and activated for the cleavage according

50 (w) cm²; H NMR (400 MHz, DMSO-d ) d 0.87 (d,

1

to Ingenito et al. After swelling the resin in THF (4 mL), a

solution of TMS–CHN (2 M in hexane) was added, and the

68

6

J¼6.8 Hz, 3H, Me), 0.91 (d, J¼6.8 Hz, 3H, Me), 1.35–1.61

2

t

(

m, 4H, CH CH ), 1.39 (s, 9H, Bu), 1.46 (s, 9H, Bu), 1.79

2

suspension was gently stirred using Ar bubbling for 2 h.

Subsequently, the resin was ﬁltered off, washed with THF

(4 mL, 4£1 min) and DMF (4 mL, 4£1 min), to prepare it

for the displacement reaction. The activated N-acylsulfo-

namide resin was swollen in DMF and ﬁltered off. A

2

m, 1H, CH(Me) ), 2.30 (m, 1H, CHCO), 2.38 (br s, 1H,

2

NH), 2.49 (m, 1H, CH), 2.99–3.10 (m, 3H, CHH, CH₂),

.23 (m, 2H, CH ), 3.35 (m, 1H, CHH), 4.17–4.28 (m, 3H,

CHCH O), 7.13–7.36 (m, 3H, 2 arom., NH), 7.41 (t,

3

2

J¼7.4 Hz, 2H, arom.), 7.68 (d, J¼7.4 Hz, 2H, arom.), 7.88

solution

of

ethyl-3-mercaptopropionate

(0.86 mL,

(

d, J¼7.5 Hz, 2H, arom.), 7.94 (br s, 1H, NH), 8.24 (br s,

6.65 mmol) and sodium thiophenolate (9 mg, 0.066 mmol)

in DMF (5 mL) was added and the resulting mixture heated

at 80 8C for 24 h. Removal of side-chain protecting groups

was accomplished in solution by treating the protected

1

3

1

DMSO-d ) d 19.5, 19.7, 20.2, 20.9, 26.1, 26.2, 26.8, 27.5,

H, NH), 11.48 (br s, 1H, COOH); C NMR (100 MHz,

6

2

1

7.9, 28.0, 31.2, 38.7, 42.6, 78.0, 82.8, 109.6, 119.9, 121.3,

27.2, 128.8, 137.3, 139.3, 142.5, 152.0, 155.1, 163.0,

b-peptide thioester with a solution of TFA/H O/TIS

2

7478

D. Seebach et al. / Tetrahedron 60 (2004) 7455–7506

(95/2.5/2.5). The solvents were removed under reduced

pressure and the precipitate, which formed upon addition of

centrifugation. The crude peptide was puriﬁed by RP-HPLC

(15–50% B in 40 min, 50–95% B in 20 min, C ) to yield

8

cold Et O to the oily residue, was collected by centrifu-

2

the TFA salt of 3 (65 mg, 38%) as a colorless ﬂuffy solid.

Homogeneity .95% (RP-HPLC). Anal. RP-HPLC: t_R

28.44 (10–40% B in 40 min, 40–95% B in 5 min, C₈).

CD (0.2 mM in MeOH, 20 8C): 213128.5 (219 nm); 0

(207.5 nm);þ14495.7 (200.5 nm); CD (0.2 mM in MeOH,

210 8C): 215643.1 (218.5 nm); 0 (206 nm);þ16894.2

(199.5 nm). ESI HRMS (positive mode): 708.3576 (100,

gation. Puriﬁcation by RP-HPLC (10–50% B in 50 min, C₈)

yielded 2 (10 mg, 10%) as a colorless ﬂuffy solid. Anal. RP-

HPLC: t_R28.34 (10–40% B in 50 min, 40–95% B in

1

0 min C ). CD (0.2 mM in MeOH, 20 8C): 211387.9

8

(

in MeOH, 210 8C): 217955.4 (216 nm);

219.5 nm); 0 (210 nm); þ9643.96 (204 nm); CD (0.2 mM

0

2

þ

2þ

(

1

(

206.5 nm);þ1895.45 (205 nm). ESI MS (positive mode):

(Mþ2Na) , C H N Na O S ; calcd 708.3625),

6

5

106 14

2

14

2þ

2

þ

2þ

C H N NaO S ;

14 2

437.9 (15, (MþH) ), 730.5 (40, (MþNaþH) ), 719.7

697.3718 (70, (MþHþNa)

,

6

5

107 14

2

þ

3þ

(Mþ2H)²^þ

100, (Mþ2H) ), 480.0 (32, (Mþ3H) ).

calcd 697.3715), 686.3795

C H N O S ; calcd 686.3805).

(10,

,

2

108 14 14 2

þ

6

5

2

(

.4.2. H-(S)b hCys-(S)b hGln-(S,S)b hIle-(S)b hLys-

2

S)b hHis-(S)b hLeu-(S)b hAsp-(S)b hMet-(S)b hPhe-

2

2.4.3. H-b hGly-(S)b hTrp-(S)b hArg-(S)b hVal-

2

S)b hPro-OH (3). Esteriﬁcation of the Wang resin was

7

(S)b hAsn-(S)b hGlu-(R,S)b hThr-(R)b hSer-

2

1

2

performed according to Chan and White. To a soln. of

2

(S)b hTyr-(S)b hAla-(S)b hCys-(S)b hGln-(S,S)

2

Fmoc-(S)b hPhe-(S)b hPro-OH (4) (461 mg, 0.9 mmol) in

dry CH Cl (4 mL) was added 1-MeIm (0.05 mL,

b hIle-(S)b hLys-(S)b hHis-(S)b hLeu-(S)b hAsp-

2

(S)b hMet-(S)b Phe-(S)b hPro-OH (1). b -Peptide frag-

2

ment 3 (5.2 mg, 3.1 mmol) and the C-terminal thioester b -

2

0

.675 mmol) followed by MSNT (267 mg, 0.90 mmol).

The mixture was stirred until all MSNT had dissolved. The

solution was then transferred to a vessel containing the

preswelled resin (200 mg, 0.18 mmol), and mixed under Ar

bubbling for 1 h at 25 8C. The resin was then ﬁltered off,

washed with DMF (4 mL, 4£1 min), CH Cl (4 mL,

peptide 2 (4.8 mg, 3.1 mmol) were ligated in an aqueous

buffer (100 mM phosphate, pH 7.5) (3 mL) containing

thiophenol (4% v/v). The ligation reaction was performed at

25 8C and monitored using anal. RP-HPLC (see Fig. 21

above). Following completion of the ligation, the reaction

2

4

£1min), and dried under h.v. overnight. The loading of

mixture was diluted with H O (1 mL) containing 0.1% TFA

2

the resin was determined by measuring the absorbance of

the benzofulvene–piperidine adduct according to Schreiber

and puriﬁed by preparative RP-HPLC (10–50% B in

40 min, 50–99% B in 10 min, C ) to yield the TFA salt of

8

0

and Seebach and was found to be 0.61 mmol/g (68%),

corresponding to 0.122 mmol of anchored 4. The unreacted

1 (5.12 mg, 54%) as a colorless ﬂuffy solid. Anal. RP-

HPLC: t_R36.82 (10–50% B in 40 min, 50–99% B in

10 min, C ). CD (0.2 mM in MeOH): 216849.8 (214 nm); 0

hydroxy groups were capped using Ac O (0.12 mL,

2

8

1

0

.22 mmol) in DMF (4 mL) and DMAP (5 mg,

.04 mmol, added in 0.5 mL DMF) for 30 min. The Fmoc

(200 nm); CD (0.2 mM in H O, pH 7): 21863.85 (220 nm);

2

0 (214.5 nm);þ15824.65 (201.5 nm). MALDI HRMS:

þ

2

group was removed using 20% piperidine in DMF (4 mL,

4

2697.428 (10, (MþNa) , C

H

N NaO S ; calcd

1

27 199 29

30

þ

£10 min) under Ar bubbling. After ﬁltration, the resin was

2697.428), 2675.442 (60, (MþH) , C₁₂₇H₁₉₉N O S ;

29

30

2

washed with DMF (4 mL, 4£1 min). Solid-phase synthesis

was continued by sequential incorporation of N-Fmoc-

protected dipeptides building blocks (5, 6, 7, 8). For each

coupling step, the resin was treated with a solution of Fmoc

building block (3 equiv.), HATU (2.9 equiv.) and DIPEA

calcd 2675.445).

Acknowledgements

(

using Ar bubbling for 45–60 min. Monitoring of the

6 equiv.) in DMF. The suspension was then gently stirred

We gratefully acknowledge generous ﬁnancial support by

ETH Z u¨ rich, the Swiss National Science Foundation,

Novartis AG, and the numerous foreign Funding Institutions

(Graduate and Postdoctoral Stipends). We thank the

2

collaborators in our group, who have prepared the b -

9

1

coupling reaction was performed with TNBS. In the

case of a positive TNBS test (indicating incomplete

coupling), the suspension was ﬁltered, and treated again

with a freshly prepared solution of the same N-Fmoc

protected building blocks (2 equiv.) and coupling reagents.

The resin was then ﬁltered off and washed with DMF (4 mL,

homoamino acids and who have done preliminary experi-

2

ments of synthesizing b -dipeptide derivatives in prep-

2

aration of the b -eicosapeptide assembly described herein:

4

£1 min) prior to the subsequent Fmoc deprotection step

Pascal Bindsch a¨ dler, Fran c¸ ois Gessier, Delphine Josien,

Gerald Lelais, Yogesh Mahajan, Peter Micuch, Laurent

Schaeffer. Part of some ﬁgures herein are reproduced by

permission of Verlag Helvetica Acta.

using 20% piperidine in DMF (4 mL, 4£10 min). After

ﬁltration, the resin was washed with DMF (4 mL, 3£1 min)

and solid-phase synthesis was continued by sequential

incorporation of N-Fmoc protected building block. For each

coupling step, the resin was treated as described above.

After the last coupling the resin was ﬁltered off, washed

with DMF (4 mL, 4£1 min), CH Cl (4 mL, 4£1 min),

2

References and notes

MeOH (4 mL, 4£1 min) and dried under h.v. for 24 h. The

dry peptide resin was treated for 2 h with a TFA/H

1. Seebach, D. Chimia 2000, 54, 751–758.

2

-

O/EDT/TIS (94:2.5:2.5:1) solution (10 mL). The resin was

removed by ﬁltration, washed with TFA, and the organic

phase containing the peptide was concentrated under

reduced pressure. The precipitate, which formed upon

2. Seebach, D. On the occasion of the 10 year anniversary

LONZA Japan on October 5, 1990, in Tokyo Nohrin Nenkin

Kaikan, PastoraleLonza Brochure, Lonza Ltd. Basel, 1990.

3. Seebach, D. Aldrichim. Acta 1992, 25, 59–66.

addition of cold Et O to the oily residue, was collected by

2

4. Seebach, D.; Geiss, K. H.; Kolb, M.; Beck, A. K. Modern

D. Seebach et al. / Tetrahedron 60 (2004) 7455–7506

7479

Synthetic Methods, Scheffold, R., Ed.; Sauerl a¨ nder, AG:

Aarau, 1976; Vol. 1, pp 173–299.

. Gfeller, J. C.; Beck, A. K.; Seebach, D. Helv. Chim. Acta 1980,

32. Seebach, D.; Fritz, M. G. Int. J. Biol. Macromol. 1999, 25,

217–236.

5

6

7

8

33. Gademann, K.; H a¨ ne, A.; Rueping, M.; Jaun, B.; Seebach, D.

Angew. Chem. 2003, 115, 1573–1575, Angew. Chem. Int. Ed.

2003, 42, 1534–1537.

6

3, 728–732.

. Seebach, D.; Bossler, H.; Gr u¨ ndler, H.; Shoda, S.-i.; Wenger,

R. Helv. Chim. Acta 1991, 74, 197–224.

. K o¨ ck, M.; Kessler, H.; Seebach, D.; Thaler, A. J. Am. Chem.

Soc. 1992, 114, 2676–2686.

34. Claridge, T. D. W.; Goodman, J. M.; Moreno, A.; Angus, D.;

Barker, S. F.; Taillefumier, C.; Watterson, M. P.; Fleet, G. W. J.

Tetrahedron Lett. 2001, 42, 4251–4255.

. Seebach, D.; Beck, A. K.; Bossler, H.; Gerber, C.; Ko, S. Y.;

Murtiashaw, C. W.; Naef, R.; Shoda, S.-i.; Thaler, A.; Krieger,

U.; Wenger, R. Helv. Chim. Acta 1993, 76, 1564–1590.

. Seebach, D.; Bossler, H.; Flowers, R.; Arnett, E. M. Helv.

Chim. Acta 1994, 77, 291–305.

35. Seebach, D.; Gademann, K.; Schreiber, J. V.; Matthews, J. L.;

Hintermann, T.; Jaun, B.; Oberer, L.; Hommel, U.; Widmer,

H. Helv. Chim. Acta 1997, 80, 2033–2038.

9

3

6. Appella, D. H.; Christianson, L. A.; Klein, D. A.; Powell,

D. R.; Huang, X. L.; Barchi, J. J.; Gellman, S. H. Nature 1997,

3

1

0. Seebach, D.; Beck, A. K.; Studer, A. Modern Synthetic

Methods, Ernst, B., Leumann, C., Eds.; VHCA Verlag

Helvetica Chimica Acta/VCH: Basel/Weinheim, 1995; Vol.

87, 381–384.

3

7. Seebach, D.; Overhand, M.; K u¨ hnle, F. N. M.; Martinoni, B.;

Oberer, L.; Hommel, U.; Widmer, H. Helv. Chim. Acta 1996,

7

, pp 1–178.

1. Miller, S. A.; Grifﬁths, S. L.; Seebach, D. Helv. Chim. Acta

993, 76, 563–595.

9, 913–941.

1

3

8. Appella, D. H.; Christianson, L. A.; Karle, I. L.; Powell, D. R.;

Gellman, S. H. J. Am. Chem. Soc. 1996, 118, 13071–13072.

9. Seebach, D.; Abele, S.; Gademann, K.; Jaun, B. Angew. Chem.

1

2. Seebach, D.; Bezen c¸ on, O.; Jaun, B.; Pietzonka, T.; Matthews,

J. L.; K u¨ hnle, F. N. M.; Schweizer, W. B. Helv. Chim. Acta

3

1

999, 111, 1700–1703, Angew. Chem. Int. Ed. 1999, 38,

595–1597.

1

996, 79, 588–608.

3. Seebach, D.; Thaler, A.; Beck, A. K. Helv. Chim. Acta 1989,

2, 857–867.

4. Thaler, A.; Seebach, D.; Cardinaux, F. Helv. Chim. Acta 1991,

4, 628–643.

1

4

0. Daura, X.; Gademann, K.; Jaun, B.; Seebach, D.; van

7

Gunsteren, W. F.; Mark, A. E. Angew. Chem. 1999, 111,

2

49–253, Angew. Chem. Int. Ed. 1999, 38, 236–240.

1. Frackenpohl, J.; Arvidsson, P. I.; Schreiber, J. V.; Seebach, D.

ChemBioChem 2001, 2, 445–455.

7

4

5. Seebach, D.; Ko, S. Y.; Kessler, H.; K o¨ ck, M.; Reggelin, M.;

Schmieder, P.; Walkinshaw, M. D.; B o¨ lsterli, J. J.; Bevec, D.

Helv. Chim. Acta 1991, 74, 1953–1990.

2. Wiegand, H.; Wirz, B.; Schweitzer, A.; Camenisch, G. P.;

Perez, M. I. R.; Gross, G.; Woessner, R.; Voges, R.;

Arvidsson, P. I.; Frackenpohl, J.; Seebach, D. Biopharm.

Drug Dispos. 2002, 23, 251–262.

1

6. B u¨ rger, H. M.; Seebach, D. Angew. Chem. 1994, 106,

4

59–460, Angew. Chem. Int. Ed. Engl. 1994, 33, 442–444.

1

7. Matt, T.; Seebach, D. Helv. Chim. Acta 1998, 81, 1845–1895.

8. Seebach, D.; Charczuk, R.; Gerber, C.; Renaud, P.; Berner, H.;

Schneider, H. Helv. Chim. Acta 1989, 72, 401–425.

4

3. Wiegand, H.; Puls, K. L.; Hoyer, D.; Kimmerlin, T.; Sebesta,

R.; Seebach, J. D.; Baumann, B. C.; Seebach, D., hitherto

unpublished results: Novartis Pharma AG, Basel, ETH Z u¨ rich

and Medical School of Z u¨ rich University.

1

2

9. Podlech, J.; Seebach, D. Angew. Chem. 1995, 107, 507–509,

Angew. Chem. Int. Ed. Engl. 1995, 34, 471–472.

0. Guibourdenche, C.; Podlech, J.; Seebach, D. Liebigs Ann.

4. Greenhow, D.; Perry, S.; Lind, R.; Kimmerlin, T.; Seebach,

D., hitherto unpublished results: Syngenta (GB Jealotts Hill)

and ETH Z u¨ rich.

1

996, 1121–1129.

1. Guibourdenche, C.; Seebach, D.; Natt, F. Helv. Chim. Acta

997, 80, 1–13.

2. Seebach, D.; Wasmuth, D. Helv. Chim. Acta 1980, 63,

97–200.

3. Seebach, D.; Aebi, J. D.; Wasmuth, D. Org. Synth. 1985, 63,

09–120.

4. Seebach, D.; Wasmuth, D. Angew. Chem. 1981, 93,

007–1008, Angew. Chem. Int. Ed. Engl. 1981, 20, 971–972.

5. Aebi, J. D.; Seebach, D. Helv. Chim. Acta 1985, 68,

507–1518.

6. Estermann, H.; Seebach, D. Helv. Chim. Acta 1988, 71,

824–1839.

4

5. Schreiber, J. V.; Frackenpohl, J.; Moser, F.; Fleischmann, T.;

Kohler, H. P.; Seebach, D. ChemBioChem 2002, 3, 424–432.

6. Gademann, K.; Kimmerlin, T.; Hoyer, D.; Seebach, D. J. Med.

Chem. 2001, 44, 2460–2468.

1

7. Nunn, C.; Rueping, M.; Langenegger, D.; Schuepbach, E.;

Kimmerlin, T.; Micuch, P.; Hurth, K.; Seebach, D.; Hoyer, D.

Naunyn-Schmiedeberg’s Arch. Pharmacol. 2003, 367,

1

9

5–103.

4

8. Seebach, D.; Namoto, K.; Mahajan, Y. R.; Bindsch a¨ dler, P.;

Sustmann, R.; Kirsch, M.; Ryder, N. S.; Weiss, M.; Sauer, M.;

Roth, C.; Werner, S.; Beer, H.-D.; Munding, C.; Walde, P.;

Voser, M. Chem. Biodiversity 2004, 1, 65–97.

1

7. Seebach, D.; Sifferlen, T.; Bierbaum, D. J.; Rueping, M.; Jaun,

B.; Schweizer, W. B.; Schaefer, J.; Mehta, A. K.; O’Connor,

R. D.; Meier, B. H.; Ernst, M.; Gl a¨ ttli, A. Helv. Chim. Acta

4

9. Rossi, F.; Lelais, G.; Seebach, D. Helv. Chim. Acta 2003, 86,

2

653–2661.

5

0. Gaul, C.; Schweizer, B. W.; Seiler, P.; Seebach, D. Helv.

Chim. Acta 2002, 85, 1546–1566.

2

002, 85, 2877–2917.

8. Seebach, D.; Beck, A. K.; Bierbaum, D. J. Chem. Biodiversity

004. in press.

9. Laube, T.; Dunitz, J. D.; Seebach, D. Helv. Chim. Acta 1985,

8, 1373–1393.

0. Seebach, D.; Sting, A. R.; Hoffmann, M. Angew. Chem. 1996,

2

3

2

51. Brenner, M.; La Vecchia, L.; Leutert, T.; Seebach, D. Org.

Synth. 2003, 80, 57–65.

6

52. Hintermann, T.; Seebach, D. Helv. Chim. Acta 1998, 81,

2093–2126.

1

08, 2880–2921, Angew. Chem. Int. Ed. Engl. 1996, 35,

708–2748.

53. Micuch, P.; Seebach, D. Helv. Chim. Acta 2002, 85,

1567–1577.

2

3

1. Seebach, D.; Boog, A.; Schweizer, W. B. Eur. J. Org. Chem.

999, 335–360.

54. Seebach, D.; Schaeffer, L.; Gessier, F.; Bindsch a¨ dler, P.;

J a¨ ger, C.; Josien, D.; Kopp, S.; Lelais, G.; Mahajan, Y. R.;

1

7

480

D. Seebach et al. / Tetrahedron 60 (2004) 7455–7506

Chem. 2003, 115, 730–736, Angew. Chem. Int. Ed. 2003, 115,

776–778.

Micuch, P.; Sebesta, R.; Schweizer, B. W. Helv. Chim. Acta

003, 86, 1852–1861.

5. Sebesta, R.; Seebach, D. Helv. Chim. Acta 2003, 86,

061–4072.

6. Lelais, G.; Campo, M. A.; Seebach, D. Helv. Chim. Acta 2004,

545–1560.

2

5

87. Arvanitis, E.; Ernst, H.; Ludwig, A. A.; Robinson, A. J.;

Wyatt, P. B. J. Chem. Soc., Perkin Trans. 1 1998, 521–528.

88. Abele, S.; V o¨ gtli, K.; Seebach, D. Helv. Chim. Acta 1999, 82,

1539–1558.

4

1

7. Lelais, G.; Micuch, P.; Josien, D.; Rossi, F.; Seebach, D. Helv.

Chim. Acta 2004. in press.

8. Kimmerlin, T.; Seebach, D.; Hilvert, D. Helv. Chim. Acta

89. Guichard, G.; Abele, S.; Seebach, D. Helv. Chim. Acta 1998,

81, 187–206.

9

0. Calbiochem-Novabiochem; Resins, S.B. S.-C. Novabiochem

Inovation 4/99, 1999.

1. Hancock, W. S.; Battersby, J. E. Anal. Biochem. 1976, 71,

2

002, 85, 1812–1826.

9

5

6

9. Kimmerlin, T.; Seebach, D. Helv. Chim. Acta 2004. in press.

0. Aurora, R.; Rose, G. D. Prot. Sci. 1998, 7, 21–38.

1. Kimmerlin, T.; Seebach, D. Helv. Chim. Acta 2003, 86,

2

60–264.

2098–2103.

6

2. Seebach, D.; Kimmerlin, T.; Jaun, B.; Mathad, R.; W u¨ thrich,

K.; Hilty, C. Unpublished results, ETH Z u¨ rich.

Article Doi

How we drifted into peptide chemistry and where we have arrived at

DOI: 10.1016/j.tet.2004.06.043

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.tet.2004.06.043

Full text of DOI:10.1016/j.tet.2004.06.043

Products guided by the article

R&D Labs maybe for 764705-06-8

Relevant to this article

Hot Product