Synthesis of β-hexa- and β-heptapeptides containing novel β2,3-amino acids with two serine or two cysteine side chains - CD- and NMR-spectroscopic evidence for 314-helical secondary structures in water

Helvetica Chimica Acta ± Vol. 83 (2000)

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Synthesis of b-Hexa- and b-Heptapeptides Containing Novel b^2,3-Amino

Acids with Two Serine or Two Cysteine Side Chains ± CD- and NMR-

Spectroscopic Evidence for 3₁₄-Helical Secondary Structures in Water

by Dieter Seebach*, Albrecht Jacobi¹), Magnus Rueping²), Karl Gademann³), Martin Ernst⁴),

and Bernhard Jaun

Laboratorium für Organische Chemie der Eidgenössischen Technischen Hochschule, ETH-Zentrum,

Universitätstrasse 16, CH-8092 Zürich

Dedicated to Albert Eschenmoser with admiration and best wishes on the occasion of his 75th birthday

Two representatives of a new type of b-amino acids, carrying two functionalized side chains, one in the 2-

and one in the 3-position, have been prepared stereoselectively: a b-Ser derivative with an additionalCH ₂OH

group in the 2-position (for b-peptides with better water solubility; Scheme 2) and a b-HCys derivative with an

additionalCH ₂SBn group in the 2-position (for disulfide formation and metal complexation with the derived b-

peptides; Scheme 3). Also, a simple method for the preparation of a-methylidene-b-amino acids is presented

(see Boc-2-methylidene-b-HLeu-OH, 8 in Scheme 3). The two amino acids with two serine or two cysteine side

chains are incorporated into a b-hexa- and two b-heptapeptides (18 and 23/24, resp.), which carry up to four

CH₂OH groups. Disulfide formation with the b-peptides carrying two CH₂SH groups generates very stable 1,2-

dithiane rings in the centre of the b-heptapeptides, and a cyclohexane analog was also prepared (cf. 27 in

Scheme 6). The CD spectra in H₂O clearly indicate the presence of 3₁₄-helical structures of those b-peptides (18,

23, 24, 27b) having the ꢀrightꢁ configurations at all stereogenic centers (Fig. 2). NMR Measurements (Tables 1

and 2, and Fig. 4) in aqueous solution of one of the new b-peptides (24) are interpreted on the assumption that

the predominant secondary structure is the 3₁₄-helix, a conformation that has been found to be typical for b-

peptides in MeOH or pyridine solution, according to our previous NMR investigations.

1. Introduction. ± b-Peptides have been shown to differ fundamentally from a-

peptides and, yet, they have made their entry as a promising class of peptidomimetics.

They fold into a variety of stable and well-ordered secondary structures like helices [1],

turns, and sheets [2]. Their large structural diversity, together with the finding that b-

peptides are not subject to degradation by mammalian peptidases [3], makes them

interesting for pharmaceutical applications. Several examples of biologically active b-

peptides have been reported in the past year [4].

In the course of our most recent structuralinvestigations, we focused on water-

solubility of b-peptides as this property is a prerequisite for in vivo function, and hence

applicability in biological systems. While water-soluble b-peptides with various degrees

of 3₁₄-helical conformation in aqueous solution were reported [5], to date the only

1

)

Postdoctoral Fellow at ETH 1998 ± 2000, partially financed by the Deutsche Forschungsgemeinschaft

(DFG).

Part of the projected Ph.D. thesis of M.R., ETH-Zürich.

Part of the Dissertation No. 13556 of K.G., ETH-Zürich, 2000. Present adress: Department of Chemistry

and ChemicalBiool gy, Harvard University, Cambridge, Massachusetts 02138.

Part of the Masterꢁs Thesis of M.E. ETH-Zürich 1998.

2

3

)

4

)

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high-resolution structural data have been obtained on a b-hexapeptide with conforma-

tional constraints on its backbone caused by ꢀunnaturalꢁ cyclohexane moieties [5c].

This approach limits the structural variability needed for the design of b-peptides that

could account for selective binding to proteins.

On the other hand, our b-peptides with proteinogenic side chains, while being

structurally more diverse, showed only low helix-forming propensities in aqueous

solution [5a,b]⁵). Based on our experience concerning the helix-stabilizing ability of a

particular b-amino-acid substitution pattern, we decided to prepare novel2,3-

disubstituted b-amino acids that should increase the helical content [1d] of b-peptides

upon their introduction into peptide sequences⁶). The b-peptides presented in this

paper were rendered water-soluble using an appropriate combination of residues with

lipophilic and functionalized side chains; ionic side chains were excluded here, as they

had been found to destabilize the 3₁₄-helical structure [5a].

2. Preparation of the b³- and of Two Novel b^2,3-Amino-Acid Derivatives for Incor-

poration into b-Peptides. ± The b³-amino-acid building blocks used in the construction

of water-soluble b-peptides were prepared starting from commercially available N-

Boc-protected a-amino acids by the Arndt-Eistert homologation procedure [1a,b].

AdditionalSer and Cys side chains were introduced into the 2-position of b³-amino

acids via alkylation of doubly lithiated derivatives to obtain the b^2,3-amino acids [8].

We first set out to prepare the l-b^2,3-disubstitued b-amino acid with two Ser side

chains. While the employment of PhCH₂OCH₂Cl in the alkylation of Boc-(R)-b³-

HSer(Bn)-OMe produced the desired derivative of the hydroxy ester 1 (Scheme 1) in

only 11% yield, the use of formaldehyde as the electrophile gave better results: a series

of experiments revealed this reaction to be highly diastereoselective when gaseous

formaldehyde in the presence of 2 equiv. of ZnBr₂was used (albeit in moderate yields).

To assign the stereochemical course of this alkylation reaction, a 1.2 :1 mixture of 1 and

the epimer 2 obtained via Claisen condensation of Boc-(R)-b³-HSer(Bn)-OMe with

HCO₂Me and subsequent borohydride reduction (Scheme 2) was debenzylated,

leading to the lactones 3 and 4 in a 1:2 ratio, together with uncyclized dihydroxy ester

5. Separation of these compounds by flash chromatography (FC) was easily achieved,

Scheme 1. Preparation of Boc-b^2,3-HSse(Bn)-OH (1) from Boc-(R)-b³-HSer(Bn)-OMe

We were nevertheless able to prepare a macrocyclic helical b³-peptide, which partially retains its secondary

structure as deduced from its CD spectra (compound 1 in [6]). Due to its poor solubility in H₂O, a high-

resolution structure of this b-peptide could be obtained only in MeOH solution [7].

Additionally, the novel b^2,3-amino acids were deviced to, possibly, act as metal-binding residues by forming

seven-membered-ring metal chelates (see below).

5

)

6

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Scheme 2. Preparation of (R,R)- and (R,S)-b^2,3-Amino-Acid Derivatives 1 and 2 and Preparation of the

Corresponding Lactones 3 and 4

Fig. 1. Stereoscopic view (ORTEP plots [9]) of the X-ray structure of 3. The structure was determined by

Dr. B. Schweizer.

and the relative configuration of both lactones 3 and 4 was established by one-

dimensionalNOE spectroscopy. Strong NOEs from the C( a)ÀH to the C(b)ÀH in 3

are compatible with a cis-relationship of these two protons; for the trans-lactone 4, only

weak NOEs were observed. Also the diastereoisomerically pure amino acid ester 2 was

converted to a lactone, the structure of which turned out to be 4, thus establishing the

relative configuration of 1 and 2. A finalproof of the configuration was provided by an

X-ray crystal-structure analysis of the cis-lactone 3 (Fig. 1).

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We then prepared the homochiral sulfur analogs, i.e., b^2,3-amino-acid derivatives of

l- or ul-configuration (reversalof CIP priority!) with one or two Cys side chains.

Enantiomerically pure Boc-(R)-b³-HCys(Bn)-OMe (6a) was obtained in 78%

overall yield from Boc-(R)-Cys(Bn)-OH on a multigram scale by the standard Arndt-

Eistert sequence. While the a-alkylation of b³-amino-acid derivatives with BnOCH₂Cl

was not successful(see above), the use of BnSCH ₂Cl/NaI as the electrophile gave a 4 :1

mixture of 7a and 2-epi-7a in 34% yield from 6a (Scheme 3). The diastereoisomers

could be separated by FC, and the major product 7a was isolated in 25% yield.

Saponification of the methyl-ester group in 7a required rather harsh conditions, which

led to partial epimerization in the product (as detected by ¹H-NMR spectroscopy). As

the method with LiOH/H₂O₂(see below) could not be applied because of possible

oxidation of the thioether group(s), we decided to use the titanate-mediated

transesterification method [10] with Me₃SiCH₂CH₂OH (TMSE) and fluoride-ion-

induced cleavage of the TMSE ester [11]. The transesterification of methyl to silylethyl

ester (7a ! 9) was carried out during 20 h at 958, and the product was obtained in

diastereoisomerically pure form (Scheme 3), and the cleavage with Bu₄NF (TBAF) in

1

THF was fast and clean to produce the acid 10 without any H-NMR-detectable

isomerization. Starting from the b-H-Leucine derivative 6b, compound 7b was

Scheme 3. Preparation of Boc-b^2,3-HCcy(Bn)₂-OH (10) and of the Substituted Acrylic-Acid Derivative 8

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prepared analogously. The selectivity of the alkylation step was comparable, and the

major diastereoisomer 7b was obtained in 23% yield after repeated FC.

Compound 7b, carrying a thioether functionality in the 2-position, was used to

prepare an acrylic-acid derivative (Scheme 3): oxidation with 1.1 equiv. NaIO₄gave the

corresponding diastereoisomeric sulfoxides (1:1 mixture, as deduced from the

¹H-NMR spectrum), and thermolysis [12] by refluxing in toluene led to essentially

quantitative formation of the methylidene derivative 8, which was purified by FC. This

method is very mild and should be applicable to b-peptides containing b-thioester side

chains, allowing for the introduction of a novel type of b-amino-acid residue in a late

step of the synthesis⁷)⁸).

3. Synthesis of the b-Peptides 18, 23, 24 (Containing the New b^2,3-Amino Acids) and

of the Cyclohexane Derivative 27. ± A maximum protection strategy with N-terminal

Boc and C-terminal MeO groups together with Bn groups for OH and SH side chains

was chosen in order to build the new b-peptides by solution synthesis⁹) and fragment

coupling through the di-, tri-, and tetrapeptide derivatives 11 ± 16, 19, and 20.

Since Bn protection of the free OH group in amino-acid derivative 1 turned out to

be problematic, we decided to leave this group unprotected for the construction of b-

peptide 18. Thus, the methyl-ester group in 1 was cleaved by LiOH/H₂O₂/H₂O in

quantitative yield ± yet some epimerization occurred. Coupling with H-b-HLeu-OMe

[1a] was carried out (with 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC)/1-

hydroxy-1H-benzotriazole (HOBt) and N-methylmorpholine)¹⁰), with the mixture of

the epimeric acids, and the mixture of diastereoisomeric dipeptides formed was

separated by repeated recrystallization to give the pure b-dipeptide ester 11 in 10%

yield. Boc Deprotection in 11 and ± sluggish ± coupling with Boc-(R)-b³-HVal-OH [1a]

gave the b-tripeptide-ester 12a in only 26% yield¹¹).

Methyl-ester hydrolysis in 12a, and Boc deprotection in 12a, followed by fragment

coupling of the resulting tripeptide derivatives 12b and 12c, gave the protected b-

hexapeptide 17 in 35% yield, which was sequentially deprotected at the C-terminus, at

the N-terminus, and at the side chains (for conditions, see Exper. Part), to provide the

target b-peptide 18 (Scheme 4). Compound 18 was purified by preparative reversed-

phase (RP) HPL chromatography to a homogeneity of > 98% and characterized by

FAB mass spectrometry (FAB-MS) and by one- and two-dimensionalNMR spectro-

scopy.

The preparation of the b-heptapeptides 23 and 24 containing a 1,2-dithiane ring in

the centralresidue (formed upon deprotection of the Cys side chains) was quite

7

)

Another possible use of 8 might be the reaction with Michael donors in order to obtain b^2,3-amino acids,

which might not be accessible through enolate alkylation.

The synthesis of b-peptides with an a-methylidene group in each and every residue is underway in our

laboratory.

This strategy had already been successfully employed in the synthesis of macrocyclic disulfide-bridged b-

peptides [6].

Although Et₃N was replaced by the weaker base NMM, 5 ± 10% epimerization occurred during the

coupling.

As severalside-products were observed in the ¹H-NMR spectrum of crude 12a, the low yield is possibly due

to competing formation of the corresponding ester by reaction with the free OH group in the amino-

hydroxy ester from Boc-deprotected 11.

8

9

10

11

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Scheme 4. Preparation of the b-Hexapeptide Derivative 17 by Fragment Coupling of 12a and 12b and

Deprotection to b-Peptide 18

straightforward (Scheme 5): the N-Boc- and S-Bn-protected b-amino acid 10 was

coupled with the tripeptides 15a [1a] and 16a, respectively, with EDC/HOBt and

NMM. b-Tetrapeptides 19 and 20 were thus obtained without any epimerization

detectable by ¹H-NMR analysis. Subsequent fragment-coupling reactions gave the fully

protected b-heptapeptide derivatives 21 (from 19 and 15b) and 22 (from 20 and 16b).

The deprotection of 21 and 22, and cyclization by disulfide formation was achieved

by a protocol that was employed before in the construction of macrocyclic disulfide-

bridged b-peptides [6]: methyl-ester saponification as the first step was quantitative in

both cases. Subsequent Na/NH₃reduction led to cleavage of the benzyl-ether and

thioether moieties in these peptides. Air oxidation of the debenzylated products under

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Scheme 5. Preparation of b-Heptapeptides 23 and 24 Containing a 1,2-Dithiane Ring in the Central Residue (5-

Amino-1,2-dithiane-4-carboxylic acid residue is abbreviated as ADTC in the text and Exper. Part)

high-dilution conditions (0.08 mm in MeOH) produced the corresponding disulfide-

bridged compounds, which were Boc-deprotected in a finalstep. Purification by

preparative RP-HPLC gave b-heptapeptides 23 and 24 in ca. 35% overall yield. While

the uniformity of 23 and 24 was established by analytical RP-HLPC (homogeneity >

98%), electrospray mass spectroscopy (ESI-MS) was used to further corroborate the

presence of the disulfide moiety in these compounds¹²). Interestingly, 23 and 24 were

not susceptible to reductive cleavage by excess dithiothreitol¹³). b-Peptide 23 was

sufficiently soluble (ca. 0.2 mm) for us to be able to obtain CD spectra in aqueous

solution, but NMR-spectroscopic characterization in H₂O was not possible. Replace-

ment of the two b³-H-Ala residues in the amino-acid sequence of 23 by two b³-H-Ser

(in 24) resulted in an at least fiftyfold increase of the solubility in H₂O (>10 mm).

12

)

Ellmanꢁs tests, which were performed on samples of 23 and 24 after preparative RP-HPLC purification,

showed that no free SH groups were present.

See Exper. Part. In contrast, various macrocyclic disulfide bridges in b-peptides could easily be cleaved

under these conditions [6].

13

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To study the CD contribution of the disulfide chromophore in 23 and 24, the

carbocyclic analogue 27b was also prepared (Scheme 6). Since the preparation of

enantiomerically pure Boc-protected trans-2-aminocyclohexanecarboxylic acid (Boc-

trans-ACHC-OH) by resolution was described as being rather capricious and not

readily reproducible [13], and since the enantioselective access to this compound

involves a multistep procedure [14], we decided to incorporate rac-Boc-trans-ACHC-

OH¹⁴) and to separate the resulting isomers in a later stage of the synthesis. We had

good reason to expect that this separation would be readily achieved, because the

diastereoisomeric b-peptides have totally different secondary structures and thereby

different properties: (1R,2R)-trans-ACHC does not fit into an (M)-3₁₄-helical

Scheme 6. Preparation of Diastereoisomeric b-Heptapeptides 27a and 27b, and Separation by Preparative

RP-HPLC

14

)

Starting from 2-aminobenzoic acid, the racemic Boc-protected trans-ACHC [13] was prepared by

dissolving-metal reduction [15] and subsequent Boc protection in 9% overall yield.

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2123

conformation which should be formed by the target b-heptapeptide containing the

ꢀcorrectꢁ (1S,2S)-trans-ACHC unit [1b][1d].

Thus, rac-Boc-trans-ACHC-OH [13][15] was coupled to the b-tripeptide ester

resulting from Boc deprotection of 15a (EDC/HOBt and NEt₃). The 1:1 mixture of b-

tetrapeptides 25a/b obtained could be separated neither by FC nor crystallization, and

was used as such in the next coupling step with the b-tripeptide acid 15b.

The fully protected diastereoisomeric b-heptapeptides 26a and 26b have indeed

drastically different solubilities: while 26a is sufficiently soluble in MeOH/CHCl₃

1

mixtures to allow for measurement of H-NMR spectra, 26a cannot be dissolved in

common organic solvents at all¹⁵). Nevertheless, we were able to saponify the 1:1

mixture of 26a/b in high yield, and, after a final Boc deprotection, the mixture 27a/27b

was analyzed by RP-HPLC, which showed that the two isomers were still present in a

1:1 ratio. Separation of 27a and 27b by preparative RP-HPLC was indeed an easy task,

as their respective retention times on a C₈column differed by almost 10 min. The

diastereoisomers 27a and 27b were thus obtained in 31 and 26% yield, respectively, and

with over 98% purity (Scheme 6).

The ¹H-NMR spectra, together with NH/ND-exchange rates in CD₃OD, gave a first

hint for the assignment of the isomers: while the half-life values t_1/2of all NH protons

were smaller than 10 min for the more polar compound 27a (t_R8.2 min), three NH

protons in compound 27b (t_R17.5 min) showed half-life values t_1/2larger than 20 h. The

reduced solvent accessibility of the amide protons is typical of stable (e.g., helical)

1

secondary and of tertiary structures [1d][16]. The large dispersion of the H-NMR

signals from the NH protons and also of the C(a) as well as the C(b) protons of 27b, as

compared to 27a, further corroborates the assignment¹⁶) of 27a as the (R,R)- and of

27b as the (S,S)-isomer.

4. Structural Characterization. ± 4.1. CD Spectroscopy. Solutions of the b-peptides

17, 18, 22, 23, 24, and 27 in MeOH and H₂O were investigated by CD spectroscopy. In

MeOH solution, a pattern of a trough at ca. 216 and a peak at ca. 198 nm is indicative of

a (M)-3₁₄-helix¹⁷). In aqueous solutions, similar CD spectra have been observed [5a],

but no independent proof of the structure of b-peptides without conformational

backbone restriction exists, so far. The situation with 23 and 24 is somewhat more

complicated, as these peptides contain disulfide chromophores that are conformation-

ally constrained in six-membered rings¹⁸). The CD spectra of the b-peptides 23, 24

(each containing 1,2-dithiane ring), and of 27 (with a cyclohexane instead the 1,2-

dithiane ring) were thus recorded in an extended range from 190 to 350 nm.

15

)

The configuration of the central b^2,3-amino-acid residue in stereoisomer-enriched samples of 26 was

deduced from a comparison with their fully deprotected analogues 27 of which the absolute configuration of

the central b^2,3-amino acid has been assigned (see below).

)

b³-Peptides forming a stable helical secondary structure usually exhibit an increased ¹H-NMR signal

dispersion in these ranges of the spectrum.

16

17

18

This was derived from numerous CD measurements and corresponding NMR structuralinvestigations of b-

peptides lacking additional chromophoric groups in the side chains [1][5a][17].

Several long- and short-wavelength CD bands have been identified for 1,2-dithiane model compounds

having an unequaldistribution of ( M)- and (P)-chiralC ÀSÀSÀC groups, that is caused by stereogenic

centers in their six-membered rings [18].

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The protected b-peptides 17 and 22 show a pattern compatible with a 3₁₄-helical

structure in MeOH solution (Fig. 2,a). Compound 17 presents the first example of a

terminally protected b-hexapeptide with proteinogenic side chains that is judged to be

Fig. 2. Overlay of CD spectra of b-peptides 17, 18, 22,

23, 24, 27a, and 27b in MeOH and H₂O. a)

Terminally Protected b-Peptides 17 and 22, b)

epimeric b-heptapeptides 27a and 27b, c) b-peptides

18 and 27b in H₂O, d) b-heptapeptides 23 and 24,

containing a disulfide chromophore, in MeOH and

H₂O, e) 23 and 24 in H₂O. Molar ellipticity [V] in 10

deg ´ cm²´ mol^À1. Al lN-deprotected b-peptides were

measured as their TFA salts (see Exper. Part).

Helvetica Chimica Acta ± Vol. 83 (2000)

2125

helical on the basis of its CD spectrum; until now, a b-heptapeptide was the shortest

terminally protected b-peptide in which the helical conformation was detectable¹⁹).

While first hints concerning the secondary structures of b-peptides 27a and 27b

1

came from one-dimensional H-NMR spectra (see above), the configuration of the

centralcycicl amino acid in the two diastereoisomers can now be assigned by

comparison of their CD spectra: whereas 27a shows no pronounced Cotton effects even

at wavelengths below 200 nm, 27b displays the typical pattern assigned previously to

the 3₁₄-helical structure with a maximum at 198 nm and a minimum at 216 nm

(Fig. 2,b)²⁰). On the basis of this analytical evidence, the configuration of the central

amino acid residue in 27a must be (R,R) and that of the isomer 27b (S,S).

The b-hexapeptide 18 shows a weaker Cotton effect at 216 nm in H₂O (Fig. 2,c).

This is in aggreement with the absence of the typical ꢀhelicalꢁ NOE crosspeaks in 2D-

NMR-spectroscopic measurements, indicating a small helical content of 18 in H₂O (see

below). b-Heptapeptide 27b, on the other hand, seems to retain a ꢀconsiderable amount

of helicityꢁ in H₂O (Fig. 2,c).

In MeOH as well as in aqueous solution the b-heptapeptides 23 and 24 show even

more intense Cotton effects than 27b (Fig. 2,d). The fact that the disulfide

chromophore in 23 and 24 contributes to the CD spectrum is quite obvious, as a new

band appears at ca. 285 nm (cf. Fig. 2,b and d), which can be assigned to an n ! s*

transition of the disulfide bond [19]. Assuming that the six-membered ring is present in

a chair conformation²¹), and further assuming that the cyclic conformer with the

substituents in equatorial positions is predominant (otherwise a helical structure could

not be formed), a (P)-chirality axis of the CÀSÀSÀC unit should be present in 23 and

24²²). The positive sign of the band at ca. 285 nm is consistent with the empirical

ꢀquadrant ruleꢁ of Carmack and Neubert [21]²³), and also with experimental data in the

literature [21 ± 24].

A very weak second band is found around 240 nm (Fig. 2,d). This band is described

in the literature as being of opposite sign compared to the longer-wavelength band and

to be very sensitive to minor perturbations and thus not very informative [25].

Concerning the CD spectra of 23 and 24 in the short-wavelength region, it must be

pointed out that the disulfide optical activity, in terms of specific structural parameters

in this region, is still not very well-understood. On the other hand, an accurate

experimental characterization below 200 nm is usually hampered by the interference of

other chromophores (e.g., amide transitions) [25].

In conclusion, it is not entirely clear to what extent the increased intensity and the

blue shift of the minimum at 208 nm in the CD spectra of 23 and 24 (compared to the

19

)

The effect of protecting groups and chain length on the stability of helical b-peptides is discussed in [17].

CD Spectra obtained with b-heptapeptides with a central b³-alanine of ꢀwrongꢁ and ꢀrightꢁ configuration (cf.

Fig. 5,a in[1b]) suggest that a helical secondary structure should not be possible for a b-heptapeptide (built

from l-b³-amino acids) with a cyclohexanecarboxylic acid of the ꢀwrongꢁ (2R,3R)-configuration in the

centralposition.

It had been shown on the basis of dipole-moment data, that 1,2-dithiane prefers a chair conformation [20].

NOE Cross-peaks as well as the respective vicinal coupling constants observed in aqueous solution support

a chair conformation of the 1,2-dithiane ring in 24 (see below).

20

21

22

)

23

)

The dependence of this lowest-energy transition on the disulfide torsion angle has also been deduced on the

basis of the Bergson model[19][22] as shown by Linderberg and Michl [23].

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carbocyclic analogue 27b) is caused by a contribution of the disulfide chromophore.

Upon switching from MeOH to H₂O, the intensity of this Cotton effect decreased by

only 10% (in comparison to a 50% decrease found for 27b), and a small blue shift is

observed. As the conformation of the 1,2-dithiane moiety should not be too dependent

on the solvent employed [20], this finding could point towards a comparatively large

disulfide contribution within this band.

It is noteworthy that the spectra of 23 and 24 are identicalwithin experimental

error, which indicates that replacing lipophilic Ala by polar Ser residues has neither a

disrupting nor a stabilizing effect on the helix.

4.2. NMR Spectroscopy of the b-Peptide 24. ± CD Measurements of peptide 24 in

aqueous solution (Fig. 2) displayed the typical 3₁₄-helical pattern. Hence, it was of interest

to determine the secondary structure of the b-peptide 24 in H₂O/D₂O in order to

ascertain whether a 3₁₄-helical conformation as shown in Fig. 3 is really present.

Therefore, this b-heptapeptide, H-(R)-b³-HVal-(R)-b³-HSer-(S)-b³-HLeu-(2S,3S)-

ADTC-(R)-b³-HVal-(R)-b³-HSer-(S)-b³-HLeuÀOH ´ CF₃CO₂H, in H₂O/D₂O was in-

1

vestigated by NMR spectroscopy. The assignment of all resonances in the H-NMR

spectrum, as well the determination of the sequence, was achieved by DQF-COSY,

TOCSY, HSQC, and HMBC experiments, and the chemicalshifts are listed in Table 1.

Fig. 3. Model of the typical 3₁₄-helical structure of hexapeptide 24

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Table 1. ¹H-NMR Chemical Shifts of the Heptapeptide 24 in H₂O

b-Amino

acid

NH

HÀC(b)

HÀC(a)

(a_la)/(a_ax

HÀC(g)

(g1)/(g2)

HÀC(g')

HÀC(d)

HÀMe

)

Val¹

Ser²

Leu³

Cys⁴

Val⁵

Ser⁶

Leu⁷

7.65

3.43

4.28

4.19

4.10

4.00

4.23

4.29

2.72/2.62

2.46/2.46

2.44/2.21

2.76

2.49/2.25

2.50/2.36

2.53/2.40

1.96

0.95/0.95

0.80/0.80

0.78

8.10 (J(NH,b) 8.9)

7.90 (J(NH,b) 9.4)

8.21 (J(NH,b) 9.0)

7.81 (J(NH,b) 9.7)

7.85 (J(NH,b) 9.5)

7.83 (J(NH,b) 9.9)

3.54/3.49

1.31/1.21

2.86/2.81

1.68

3.47/3.47

1.36/1.30

1.45

1.50

3.07/3.07

0.97/0.97

The NH protons of residues 2 ± 7 show coupling constants J(NH,H ± C(b)) in the

range of 9 ± 10 Hz, which corresponds to an antiperiplanar arrangement of the NH and

HÀC(b). The assignments of the diastereotopic CH₂(a) protons were made on the

assumption that the axialprotons, H _axÀC(a) exhibit a large and the lateral protons,

1

H_laÀC(a) a small coupling with HÀC(b) as evident from H-NMR and DQF-COSY

spectra. This is in agreement with the observation that the HÀC(b) show stronger

NOE to the H_laÀC(a), the proton with the smaller vicinal coupling constant, than to

H_axÀC(a). To gather more information about the three-dimensionalstructure,

ROESY spectra at different mixing times were acquired, and NOEs were extracted

from the ROESY spectra with a mixing time of 150 ms. A totalof 96 NOEs were

extracted and classified according to their cross-peak volume into three distance

categories: strong, medium, and weak (Table 2).

Table 2. Weak (w, 4.5 ), Medium (m, 3.5 ), and Strong (s, 3.0 ) NOEs Observed in the ROESY NMR

Spectrum of Compound 24 in H₂O

Residue

H-Atom(s)

Residue

H-Atom(s)

NOE

1

2

NH

b

1

2

4

5

2

1

2

1

3

2

3

Me

g

m

w

m

w

m

w

m

w

m

w

m

s

b

a_ax

g

a_1a

Me

g

a_1a

a_ax

Me

g

a_ax

NH

b

g1

g2

a_1a

a_ax

a

m

w

Me

a_1a

g1

b

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Helvetica Chimica Acta ± Vol. 83 (2000)

Table 2 (cont.)

Residue

H-Atom(s)

Residue

H-Atom(s)

NOE

2

3

4

5

b

3

2

3

5

6

2

3

4

3

4

1

4

3

4

5

4

5

2

5

Me

a

m

w

s

g1

g2

a

NH

b

g

a

a_ax

d

s

m

w

m

w

m

w

m

s

g1

g2

Me

a_ax

g1

g2

Me

g2

g1

d

b

a_1a

a_ax

NH

b

Me

g2

g1

b

g1

g2

a

a_1a

a_ax

ga

Me

a_1a

Me

b

m

s

m

w

m

w

m

s

w

m

w

m

w

m

w

m

b

g'

b

g'

g1

g2

a

NH

b

g'

a

a_1a

a_ax

g

Me

a_1a

a_ax

g

b

Me

g

a_1a

a_ax

Me

Helvetica Chimica Acta ± Vol. 83 (2000)

2129

Table 2 (cont.)

Residue

H-Atom(s)

Residue

H-Atom(s)

NOE

5

6

7

a_ax

NH

b

5

6

5

6

5

6

5

7

6

4

7

g

w

m

w

m

w

s

b

g

a_ax

a_1a

a_ax

g

b

a_1a

a_ax

Me

b

g

a_1a

NH

b

a_1a

a_ax

a_1a

a_ax

d

s

w

m

w

m

w

a_ax

a

b

a_1a

d

a_1a

g1

Qualitative inspection reveals that NOEs typical for the 3₁₄-helix (Fig. 3) are indeed

found in the ROESY spectra of 24 in H₂O/D₂O 9 :1, but some of these NOEs are

considerably weaker than those observed for 3₁₄-helical b-peptidic structures in organic

solvents [1].

Inside the 3₁₄-helix, each NH_itypically exhibits two strong sequential NOEs to the

H_axÀC(a) protons of residues i À 1 and i. Structurally more significant, however, are

the NOEs of medium strength of NH_ito HÀC(b)_i2(and HÀC(b)_i3and between

H_axÀC(a)_iand HÀC(b)_i2which connect non sequentialresidues ( Fig. 4).

Compared to heptapeptide H-b-HVal-b-HAla-b-HLeu-b-HAla(aMe)-b-HVal-b-

HAla-b-HLeu-OH in CD₃OH [1d,e], which forms the most stable 3₁₄-helix, we have

observed so far, the ratio between nonsequential and sequential NOEs is much smaller

for heptapeptide 24 in H₂O. This result would be consistent with either a major

conformation with a more extended helix or, more plausibly, with the population of

both 3₁₄-helical and nonhelical or at least partially unwound conformations [1e][17].

We gratefully acknowledge the financial support of the Deutsche Forschungsgemeinschaft (DFG)

(fellowship No. JA 918/1-1 to A. J.). We are thankfulfor the assistance by the staff in our anayl ticaldivision:

B. Brandenberg and P. Zumbrunnen (NMR), Dr. W. Amrein, O. Greter, and R. Häfliger (MS) and D. Manser

(elemental analysis). Continuing support from Novartis Pharma and Agro AG (Basel) is appreciated.

2130

Helvetica Chimica Acta ± Vol. 83 (2000)

Fig. 4. Schematic NOE pattern observed for 3₁₄-helical conformations in MeOH (left). NH_ishows strong NOEs

to protons H_axÀC(a)_iÀ1,iand NOEs of medium strength to HÀC(b)_i2and HÀC(b)_i3, and between H_axÀC(a)_i

and HÀC(b)_i2, compared to NOEs observed in H₂O for heptapeptide 24 (right), where NOEs between NH

and HÀC(b) are weaker and NOEs from H_axÀC(a)_ito HÀC(b)_i2are partially not observed.

Experimental Part

1. General. Abbreviations: Boc₂O: di(tert-butyl) dicarbonate, DME: 1,2-dimethoxyethane, DTT: dithio-

threitol, EDC: 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride, FC: flash chromatography,

HOBt: 1-hydroxy-1H-benzotriazole, h.v.: high vacuum (0.01 ± 0.1 Torr), LDA: lithium diisopropylamide,

NMM: N-methylmorpholine. Solvents for chromatography and workup procedures were distilled from Sikkon

(anh. CaSO₄; Fluka). Et₃N and NMM were distilled from CaH₂and stored over KOH. ClCO₂Et was distilled

and stored at 48 (refrigerator) under Ar. DME was distilled over Na. NaI was dried under h.v. at 1508 for 16 h.

BuLi was used as a 1.5m soln. in hexane. All indicated temp. were monitored with an internal thermometer

(Ebro-TTX-690 digitalthermometer). Amino-acid derivatives were purchased from Bachem, Degussa, or Senn.

All other reagents were used as received from Fluka. The b-amino-acid derivatives Boc-(R)-b³-HVal-OH [1a],

Boc-(R)-b³-HSer(Bn)-OH [6], and Boc-(S)-b³-HLeu-OMe [1a], and the peptide derivatives Boc-(R)-b³-HVal-

(R)-b³-HAla-(S)-b³-HLeu-OMe (15a) [1a] and Boc-(R)-b³-HVal-(R)-b³-HAla-(S)-b³-HLeu-OH (15b) [1a],

and trans-Boc-ACHC-OH [13][15] were prepared according to literature procedures. BnSCH₂Clwas prepared

according to literature procedure [26]. Reactions carried out with the exclusion of light were performed in flasks

completely wrapped in aluminium foil. TLC: Merck silica gel 60 F₂₅₄plates; detection with UVand anisaldehyde

or I₂. FC: Fluka silica gel 60 (40 ± 63mm); at ca. 0.2 bar. Anal. HPLC: Knauer HPLC system K 1000,

EuroChrom 2000 Integration Package, degasser, UV detector K 2000 (variable-wavelength monitor), Macher-

ey-Nagel C₁₈column: Nucleosil 100-5 C₁₈(250 Â 4 mm). Prep. HPLC: Knauer HPLC system: pump type 64,

programmer 50, UV detector (variable-wavelength monitor); Macherey-Nagel C₁₈column: Nucleosil 100-7 C₁₈

(250 Â 21 mm). M.p. Büchi-510 apparatus; uncorrected. Opticalrotations: Perkin-Elmer 241 polarimeter

(10 cm, 1 ml cell) at r.t. Circular dichroism (CD) spectra: Jasco J-710, recording from 190 to 250 nm at r.t.; 1 mm

rectangular cell; average of five scans, corrected for the baseline; peptide concentration 0.2 mm in MeOH or

H₂O; molar ellipticity V in deg ´ cm²´ dmol^À1(l in nm); smoothing by Jasco software. IR Spectra: Perkin-Elmer-

782 spectrophotometer. NMR Spectra: Bruker AMX 500 (¹H: 500 MHz, ¹³C: 125 MHz), AMX 400 (¹H:

400 MHz, ¹³C: 100 MHz), Varian Gemini 300 (¹H: 300 MHz, ¹³C: 75 MHz); chemicalshifts d in ppm downfield

from internalMe Si (0 ppm); J values in Hz. Mass Spectra: VG Tribrid (EI), VG ZAB2-SEQ (FAB, in a 3-

4

nitrobenzyl-alcohol (3-NOBA) matrix), and Finnigan TSQ 7000 (ESI, sprayed from a 10^À5m MeOH/soln.;

volumetric flow 20 ml/min) spectrometer; in m/z (% of basis peak). Elemental analyses were performed by the

Microanalytical Laboratory of the Laboratorium für Organische Chemie, ETH-Zürich.

2. Boc Deprotection: General Procedure 1 (GP 1). Similarly to the reported procedure [1], the Boc-

protected amino acid was dissolved in CH₂Cl₂(0.5m) and cooled to 08. An equalvoul me of CF ₃CO₂H was

added, and the mixture was allowed to slowly warm to r.t. and then stirred for further 1.5 h. Concentration under

reduced pressure and drying of the residue under h.v. (12 h) yielded the crude CF₃CO₂H salt, which was

identified by NMR and used without further purification.

Helvetica Chimica Acta ± Vol. 83 (2000)

2131

3. Methyl-Ester Hydrolysis: General Procedures 2 (GP 2). a) The fully protected oligopeptide was dissolved

in MeOH (0.1m) and treated with 1n NaOH (1.2 equiv.). After 24 h, the mixture was diluted with H₂O, and the

pH was adjusted to 2 ± 3 with 1n HCl. The soln. was extracted with AcOEt, and the combined org. phases were

washed successively with sat. aq. NaCl soln. and H₂O. The org. phase was evaporated and dried under h.v.

b) The fully protected oligopeptide was dissolved in CF₃CH₂OH (0.1m) and treated with 5n NaOH

(100 equiv.) and stirred at 458. After completion of the reaction (TLC), the mixture was diluted with H₂O, and the

pH was adjusted to 2 ± 3 with 5n HCl(0 8). The soln. was extracted with AcOEt, and the combined org. phases

were washed successively with sat. NaCl soln. and H₂O. The org. phase was evaporated and dried under h.v.

4. Peptide Coupling with EDC: General Procedures 3 (GP 3). a) The appropriate CF₃CO₂H salt was

dissolved in CHCl₃(0.5m) and cooled to 08. This was treated successively with Et₃N (5 equiv.), HOBt

(1.2 equiv.), a soln. of the Boc-protected fragment (1 equiv.) in CHCl₃(0.5m), and EDC (1.2 equiv.). The

mixture was allowed to warm to r.t. and then stirred for 18 h. Subsequent dilution with CHCl₃was followed by

thorough washing with 1n HCl, sat. aq. NaHCO₃soln., and sat. aq. NaCl soln. The org. phase was dried (MgSO₄)

and then concentrated under reduced pressure. FC yielded the pure peptide.

b) The appropriate CF₃CO₂H salt was dissolved in CHCl₃(0.5m) and cooled to 08. This was treated

successively with Et₃N (5 equiv.), HOBt (1.2 equiv.), a soln. of the Boc-protected fragment (1 equiv.) in DMF

(0.1m), and EDC (1.2 equiv.). The mixture was allowed to warm to r.t. and then stirred for 18 h. The solvents

were removed under reduced pressure, and the residue was dispersed in CHCl₃(0.02m). The resulting

suspension was washed with 1m HCl, sat. aq. NaHCO₃soln., sat. aq. NaCl soln., and H₂O. The org. phase was

concentrated under reduced pressure. FC or recrystallization yielded the pure peptide.

5. Cleavage of S-Benzyl Groups/O-Benzyl Groups and Cyclization to the Disulfide: General Procedure 4

(GP 4). According to [3], the Boc-protected oligopeptide derivative was dissolved in NH₃(2 mm) at À338. An

amount of 5 equiv. of Na per Bn group was added. The dark blue soln. was stirred for 2 h (TLC) and

subsequently quenched by addition of MeOH. The resulting MeOH soln. of NH₃was immediately diluted to a

peptide concentration of 0.08 mm. Air was bubbled through the soln. for 12 h with slow stirring. MeOH was

removed under reduced pressure, and the resulting crude cyclic peptide was Boc-deprotected according to GP 1.

6. Reversed-Phase (RP) HPLC Analysis and Purification of b-Peptides: General Procedure 5 (GP 5). RP-

HPLC Analysis was performed on a Macherey-Nagel C₁₈column/Nucleosil 100-5 C₁₈(250 Â 4 mm) with a linear

gradient of A (0.1% CF₃CO₂H in H₂O) and B (MeCN) at a flow rate of 1 ml/min (UV detection at 220 nm); t_R

in min. Crude products were purified by prep. RP-HPLC (Macherey-Nagel C₁₈column/Nucleosil 100-7 C₁₈

(250 Â 21 mm) with a gradient of A and B at a flow rate of 20 ml/min (UV detection at 220 nm) and then

lyophilized.

7. Attempted Ring Opening of 23 and 24. A 0.2 mm MeOH soln. of the respective peptide was purged with

Ar and treated with 50 equiv. KOH and 25 equiv. DTTunder Ar. The reaction was monitored by RP-HPLC. No hint

for the formation of ring opened species in the respective reaction solns. of 23 and 24 was obtained during 2 d.

8. NMR Spectroscopy of the Heptaptide 24. Sample: 8 mg dissolved in 0.6 ml H₂O/D₂O. 1D-NMR

(AMX500): ¹H-NMR (500 MHz): suppression of the D₂O signalby presaturation; 90-K data points, 256 scans,

5.6-s acquisition time. {¹H}-BB-Decoupled-¹H-NMR (125 MHz): 80-K data points, 25-K scans, 1.3-s acquisition

time, 1-s relax. delay 458 excitation pulse. Processed with 1.8-Hz exponential line broadening. 2D-NMR. All

with solvent suppression by presat. DQF.COSY (500 MHz, D₂O) with pulsed field gradients (PFG) for

coherence pathway selection [27]: Acquisition: 2K(t₂) Â 512 (t₁) data points. 8 scans per t₁increment, 0.22-s

acquisition time in t₂; relaxation delay 2.0 s. TPPI Quadrature detection in w₁. Processing: Zero filling and FT to

1K Â 1K real/real data points after multiplication with sin²filter shifted by p/3 in w₂and p/2 in w₁. HSQC with

PFG [28] (500, 125 MHz, D₂O): Acquisition: 2K(t₂) Â 512 (t₁) data points, 48 scans per t₁increment. ¹³C-GARP

Decoupling during t₂, 0.22-s acquisition time in t₂. Processing: Zero filling and FT to 1K Â 1K real/real data

points after multiplication with sin²filter shifted by p/2 in w₂and sin filter shifted by p/2 in w₁. HMBC with PFG

[29] (500, 125 MHz, D₂O): Acquisition: Delay for evolution of long-range antiphase magn. 50 ms. No ¹³C-

decoupling, otherwise identical to parameters for HSQC. Processing: Zero filling and FT to 1K Â 1K after

multiplication with cos²filter in w₂and gaussian filter in w₁; power spectrum in both dimensions. ROESY [30]

(500 MHz, D₂O; see Table 2). Acquisition: 2 ROESY Spectra with mixing times of 80 and 150 ms were

acquired. CW-spin lock (3.8 kHz) between trim pulses, 2K(t₂) Â 480 (t₁) data points, 64 scans per t₁increment.

0.22-s acquisition time in t₂, other parameters identicalto DQF.COSY. Processing: Zero filling and FT to 1K Â

512K real/real data points after multiplication by cos²filter in w₂and w₁. Baseline correction with 3rd degree

polynomial in both dimensions.

Methyl

(2R,3R)-4-(Benzyloxy)-3-{[(tert-butoxy)carbonyl]amino}-2-(hydroxymethyl)butanoate

(1).

(i-Pr)₂NH (2.2 ml) was dissolved in 40 ml of THF and cooled to À258 under Ar. BuLi (10.5 ml, 1.78m in

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Helvetica Chimica Acta ± Vol. 83 (2000)

hexane, freshly titrated) was added under stirring, and the resulting soln. was cooled after 30 min to À788. Boc-

b³-HSer(OBn)-OMe (2.3 g, 7.1 mmol) were dissolved in a separate flask in 12 ml of THF and cooled to À788.

This soln. was added via syringe to the LDA soln. at À788, and the resulting suspension was stirred for 30 min. In

another flask, a suspension of ZnBr₂(3.2 g, 14.2 mmol) in 8 ml of THF was cooled to À788, stirred for 10 min,

and added to the enolate. The resulting soln. was stirred for additional 30 min. Paraformaldehyde was heated to

ca. 1808 (heatgun) in a different flask, and the resulting HCHO was transported in an Ar stream over the

reaction soln. at À788 for 15 min. After stirring the mixture for additional40 min, the reaction was stopped by

the addition of sat. NH₄Clsoln., Et ₂O was added, and the org. phase was washed with Na₂SO₄soln. and sat. NaCl

soln. The org. phase was dried (MgSO₄), and the solvent was removed under reduced pressure. FC (Et₂O/

pentane) gave 0.6 g (1.7 mmol, 24%) of diastereoisomerically pure 1. Colorless oil. R_f0.32 (Et₂O/pentane

1.5 : 1). [a]_D 5.0 (c 1.04, CHCl₃).IR (CHCl₃): 3684w, 3618w, 3438w, 3008s, 2976m, 2434w, 1723m, 1505s,

1436m, 1393m, 1368m, 1248s, 1165s, 1046s, 929m, 877w, 850w, 636m. ¹H-NMR (300 MHz, CDCl₃): 1.45 (s, t-Bu);

2.90 ± 2.97 (m, CHC(O)O); 3.56 ± 3.84 (m, MeO, CH₂O); 4.20 ± 4.26 (m, CHN); 4.50 (s, PhCH₂); 5.35 (d, J 9.7,

BocNH); 7.28 ± 7.38 (m, 5 arom. H). ¹³C-NMR (300 MHz, CDCl₃): 28.30; 48.01; 49.28; 51.69; 60.69; 70.95; 73.31;

80.25; 127.65; 127.79; 128.41; 137.64; 159.81; 172.88. FAB-MS: 729.3 (43.8, [2M Na] ), 376.1 (31.1, [M Na] ),

354.15 (45.4, [M H] ). Anal. calc. for C₁₈H₂₇NO₆(353.41): C 61.17, H 7.70, N 3.96; found: C 61.00, H 7.60, N 3.98.

Methyl

(2S,3R)-4-(Benzyloxy)-3-{[(tert-butoxy)carbonyl]amino}-2-(hydroxymethyl)butanoate

(2).

(i-Pr)₂NH (0.61 ml) was dissolved in 11 ml of THF and cooled to À258 under Ar. BuLi (2.95 ml, 1.78m in

hexane, freshly titrated) was added under stirring, and the resulting soln. was cooled after 30 min to À788. Boc-

b³-HSer(OBn)-OMe (650 mg, 2 mmol) was dissolved in a separate flask in 3.4 ml of THF and cooled to À788.

This soln. was added via syringe to the LDA soln. at À788, and the resulting suspension was stirred for 30 min.

HCO₂Me (0.25 ml) was added to the enolate, and the mixture was stirred for 1 h at À708. After stirring the

mixture for additional40 min, the reaction was stopped by the addition of sat. NH ₄Clsoln., Et ₂O was added, and

the org. phase was washed with Na₂SO₄and sat. NaCl soln. The combined org. layers were dried (MgSO₄) and

evaporated. The resulting b-amino b-oxo acid ester was dissolved in 2 ml of EtOH, and NaBH₄(31 mg,

0.8 mmol) was added. The mixture was stirred for 1 h, and the solvent was evaporated. The residue was taken up

in Et₂O, and washed with Na₂SO₄and sat. NaCl solns. The combined org. layers were dried (MgSO₄) and

evaporated to give a mixture of the diastereoisomers 1 and 2 (dr 1.2 :1; 326 mg; 46%). This mixture was

separated by prep. HPLC on ChiraSpher (hexane/i-PrOH 95 :5) to give 1 (154 mg; 22%) and 2 (129 mg; 28%).

Colorless oil. R_f0.32 (Et₂O/pentane 1.5 :1). [a]_D 8.0 (c 1.03, CHCl₃). IR (CHCl₃): 3438m, 3008m, 2980m,

1

1729s, 1503s, 1455m, 1437m, 1393w, 1368m, 1329m, 1169s, 1129w, 1082m, 862w. H-NMR (300 MHz, CDCl₃):

1.45 (s, t-Bu); 2.76 (dt, J 3.1, 9.4, CHC(O)O); 3.58 ± 3.68 (m, MeO, CH₂O); 3.78 ± 3.96 (m, CH₂O); 4.21 ± 4.30

(m, CHN); v_A 4.46, v_B 4.52 (AB, J_AB 11.8, PhCH₂O); 5.25 (d, J 9.0, BocNH); 7.26 ± 7.38 (m, 5 arom. H).

¹³C-NMR (300 MHz, CDCl₃): 28.30, 48.46; 48.57; 51.84; 60.98; 70.40; 73.32; 80.26, 127.76; 127.89; 128.45; 137.59;

157.50; 173.05. FAB-MS: 730.0 (3.9, [2M Na] ), 376.5 (16.5, [M Na] ), 354.5 (21.5, [M H] ). Anal. calc.

for C₁₈H₂₇NO₆(353.41): C 61.17, H 7.70, N 3.96; found: C 61.14, H 7.57, N 3.98.

tert-Butyl N-[(3R,4R)-2,3,4,5-Tetrahydro-4-(hydroxymethyl)-5-oxofuran-3-yl]carbamate (3) and tert-Bu-

tyl N-[(3R,4S)-2,3,4,5-Tetrahydro-4-(hydroxymethyl)-5-oxofuran-3-yl]carbamate (4). A mixture of 1 and 2

(1.02 g, 2.8 mmol) was treated with 300 mg of Pd/C (10%) in 7 ml of MeOH and 0.5 ml of AcOH for 48 h under

an H₂atmosphere. Filtration over Celite and FC (1. Et₂O/Pentane 3 :1 to Et₂O and 2. CH₂Cl₂/MeOH 15 :1) gave

3 (50 mg, 8%) and 4 (106 mg, 14%).

Data for 3: Colorless crystals. ¹H-NMR (300 MHz, CDCl₃): 1.46 (s, t-Bu); 2.88 ± 2.93 (m, CHCO)); 3.94 ±

4.07 (m, CH₂OH); 4.24 (dd, J 2.5, 10.0, 1 H, CH₂O); 4.44 (dd, J 6.3, 10.0, 1 H, CH₂O); 4.62 ± 4.66 (m, CHN);

5.61 (br., BocNH). ¹³C-NMR (300 MHz, CDCl₃): 28.27; 45.75; 50.29; 58.25; 72.87; 80.81;156.25; 175.59. NOE:

Irrad. at 4.64 ppm, strong pos. NOE at 2.90 ppm. X-Ray crystalstructure of 3: from a crystalof size 0.35 Â 0.3 Â

0.2 mm 1251 reflections were measured on an Enraf Nonius CAD-4 Diffractometer with CuK_aradiation

(graphite monochromator, l 1.54184 ). The structure was solved by direct method with SHELXS-96 [31].

The non-H-atoms were refined anisotropically with SHELXL-97 [32]. H-Atoms were obtained from a

difference Fourier map and refined isotropically. Drawings of the molecule were done with PLUTO, ORTEP

[33]. For final R values and experimental data, see Table 3.

Data for 4: Colorless crystals. R_f0.17 (Et₂O/pentane 3 :1). M.p. 114 ± 1158. [a]_D 13.2 (c 0.50, CHCl₃).

IR (CHCl₃): 3437w, 2980w, 1776s, 1709s, 1503m, 1393w, 1369m, 1162s, 1035w. ¹H-NMR (300 MHz, CDCl₃): 1.46

(s, t-Bu); 2.65 ± 2.71 (m, CHCO)); 3.95 ± 4.08 (m, 1 H, CH₂O); 4.36 ± 4.47 (m, CHN); 4.59 (dd, J 7.8, 8.7, 1 H,

CH₂O); 4.81 (br., BocNH). ¹³C-NMR (300 MHz, CDCl₃): 28.27; 48.89; 50.25; 60.09; 70.04; 81.09; 156.51;

175.00. NOE: Irrad. at 4.40 ppm, weak NOE at 4.00 ppm. EI-MS: 232.1 (0.4, [M H] ), 158.1 (79.6, [M À t-

Helvetica Chimica Acta ± Vol. 83 (2000)

2133

Table 3. Crystallographic Data and Structure Refinement for Compound 3

Crystallized from

Empiricalformual

Formula weight

Temp.

Et₂O/pentane

C

₁₀H₁₇NO₅

231.25

293(2) K

Wavelength

1.54184

Crystalsystem

Space group

Orthorhombic

P2₁2₁2₁

Unit-cell dimensions

a 10.411(3) a 908

b 12.055(2) b 908

c 9.534(6)

1196.6(9) A³

4

g 908

V

Z

Density (calc.)

Absorption coefficient

F(000)

1.284 Mg/m³

0.870 mm^À1

496

Crystalsize

q Range for data collection

Index ranges

0.35 Â 0.3 Â 0.2 mm

5.61 to 66.918

0 ꢀ h ꢀ 12, 0 ꢀ k ꢀ 14, 0 ꢀ l ꢀ 11

1251

1245 (R(int) 0.0000)

Full-matrix least-squares on F²

1216/0/213

Reflections collected

Independent reflections

Refinement method

Data/restraints/parameters

Goodness-of-fit on F²

Final R indices [I > 3s(I)]

R Indices (all data)

Absolute structure parameter

Extinction coefficient

Largest diff. peak and hole

1.523

R₁ 0.0418, wR₂ 0.1426

R₁ 0.0423, wR₂ 0.1441

0.7(3)

0.021(4)

0.204 and À0.301 eA^À3

Bu H] ), 131.1 (26.7, [M À Boc H] ), 57.0 (100, [t-Bu] ). Anal. calc. for C₁₀H₁₇NO₅(231.25): C 51.94, H 7.41,

N 6.06; found: C 51.93, H 7.39, N 6.06.

Methyl (3R)-4-(Benzylsulfanyl)-3-{[(tert-butoxy)carbonyl]amino}butanoate (Boc-(R)-b³-HCys(Bn)-OMe;

6a). Treatment of a soln. of Boc-(S)-Cys(Bn)-CHN₂(8.0 g, 23.9 mmol) according to [1] followed by workup

gave 6a as a yellow oil. FC (cyclohexane/AcOEt 4 :1) yielded pure 6a (7.56 g, 94%) in the form of colorless

crystals. M.p. 61 ± 628. R_f0.55 (cyclohexane/AcOEt 4 :1). [a]^r_D^:t: 5.9 (c 1.0, CHCl₃). IR (KBr): 3349s,

2965m, 1734s, 1681s, 1534s, 1433m, 1389w, 1370m, 1352m, 1303m, 1275m, 1207m, 1159s, 1053m, 1028m, 987w,

706s, 652m. ¹H-NMR (400 MHz, CDCl₃): 1.45 (s, t-Bu); 2.56 ± 2.72 (m, CH₂O, CH₂S); 3.66 (s, MeO); 3.73

(s, PhCH₂S); 4.06 ± 4.14 (m, CHN); 5.11 (br., NH); 7.22 ± 7.34 (m, 5 arom. H). ¹³C-NMR (100 MHz, CDCl₃):

28.4 (Me); 35.2, 36.3, 37.3 (CH₂); 46.8 (CH); 51.8 (Me); 79.6 (C); 127.1, 127.5, 128.5, 129.0 (arom. C); 138.0,

155.1, 171.8 (C). FAB-MS: 679 (25, [2M 1] ), 362 (11, [M Na] ), 340 (94, [M 1] ), 284 (100, [M À C₄H₈

1] ), 240 (91, [M À Boc 1] ). Anal. calc. for C₁₇H₂₅NO₄S₂(339.45): C 60.15, H 7.42, N 4.13, S 9.45; found:

C 60.30, H 7.42, N 4.19, S 9.51.

Methyl (2S,3R)-4-(Benzylsulfanyl)-2-[(benzylsulfanyl)methyl]-3-{[(tert-butoxy)carbonyl]amino}butanoate

(Boc-(2S,3R)-HCcy(Bn)₂-OMe; 7a). NaI (1.5 g, 10 mmol) was dissolved in 6 ml of DME. BnSCH₂Cl(1.72 g,

10 mmol) was added, and the soln. was stirred for 30 min at r.t. In a second flask, (i-Pr)₂NH (1.22 g, 12 mmol)

was dissolved in 7 ml of THF and cooled to À788. BuLi (7.5 ml, 12 mmol) was added during 10 min. Compound

6a (1.7 g, 5 mmol) was dissolved in 12 ml of THFand added over 20 min to the (i-Pr)₂NH soln. After 30 min, the

cooled (À 788) soln. of BnSCH₂Clwas added via a Teflon cannula during 10 min, and the mixture was allowed to

reach 08 over 14 h. The mixture was quenched with sat. NH₄Cl soln., diluted with Et₂O, and extracted with

NaHCO₃, NH₄Cl, and NaCl solns. The org. phase was dried (MgSO₄) and then concentrated under reduced

pressure to yield a crude epimeric mixture (803 mg; 34%; 7a/epi-7a 4 :1). FC (cyclohexane/AcOEt 6 :1) yielded

pure 7a (583 mg, 25%). White solid. M.p. 79 ± 808. R_f0.65 (cyclohexane/AcOEt 6 :1). [a]^r_D^:t: À7.0 (c 1.0,

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Helvetica Chimica Acta ± Vol. 83 (2000)

CHCl₃). IR (KBr): 3382m, 2962m, 1735s, 1677s, 1509s, 1262m, 1165m, 1090s, 1024s, 858w, 801s, 699m. ¹H-NMR

(500 MHz, CDCl₃): 1.45 (s, t-Bu); 2.36 ± 2.40, 2.51 ± 2.56, 2.72 ± 2.77 (3m, 2 CH₂S); 3.04 ± 3.08 (m, CHCO); 3.67

(s, MeO); 3.72, 3.75 (2s, 2 CH₂); 4.06 ± 4.14 (m, CHN); 5.32 (br., NH); 7.22 ± 7.35 (m, 10 arom. H). ¹³C-NMR

(125 MHz, CDCl₃): 28.4 (Me); 30.8, 34.7, 36.0, 36.4 (CH₂); 46.6, 50.1 (CH); 52.0 (Me); 79.6 (C); 127.1; 127.2,

128.5, 128.6, 129.0, 129.1 (arom. C); 137.8, 137.9, 155.5, 173.6 (C). FAB-MS: 476 (15, [M 1] ), 420 (24), 385

(46, [M À Bn 1] ), 376 (100, [M À Boc 1] ), 328 (66), 296 (29). Anal. calc. for C₂₅H₃₃NO₄S₂(475.67):

C 63.13, H 6.99, N 2.94, S 13.48; found: C 63.17, H 6.82, N 2.84, S 13.42.

Methyl (2S,3S)-2-[(Benzylsulfanyl)methyl]-3-{[(tert-butoxy)carbonyl]amino}-5-methylhexanoate (7b).

NaI (1.5 g, 10 mmol) was dissolved in 6 ml of DME. BnSCH₂Cl(1.72 g, 10 mmo)l was added, and the sonl .

was stirred for 30 min at r.t. In a second flask, (i-Pr)₂NH (1.22 g, 12 mmol) was dissolved in 7 ml of THF and

cooled to À788. BuLi (7.5 ml, 12 mmol) was added during 10 min. Boc-(S)-b³-HLeu-OMe (777 mg, 3 mmol)

was dissolved in 12 ml of THF and added over 20 min to the (i-Pr)₂NH soln. After 30 min, the cooled (À 788)

soln. of BnSCH₂Clwas added via a Teflon cannula during 10 min, and the mixture was allowed to reach 08 over

14 h. The mixture was quenched with sat. NH₄Cl soln., diluted with Et₂O, and extracted with NaHCO₃, NH₄Cl,

and NaClsonl s. The org. phase was dried (MgSO ₄) and then concentrated under reduced pressure yielding a

crude epimeric mixture (681 mg, 57%; 7b/epi-7b 5 :1). FC (cyclohexane/AcOEt 6 :1) yielded pure 7b (268 mg,

23%). White solid. M.p. 43 ± 448. R_f0.6 (cyclohexane/AcOEt 6 :1). [a]^r_D^:t: À6.9 (c 1.0, CHCl₃). IR (KBr):

3392m, 2956m, 1719s, 1503s, 1454m, 1437m, 1366m, 1246m, 1206m, 1164s, 1111w, 1022w, 805w, 702m. ¹H-NMR

(400 MHz, CDCl₃): 0.88 (d, J 6.5, Me); 0.89 (d, J 6.0, Me); 1.11 ± 1.30 (m, 2 H, 2 CH₂); 1.42 (s, t-Bu); 1.58 ±

1.63 (m, CH); 2.56 ± 2.77 (m, CH₂S, CHCO); 3.71 (br., MeO, PhCH₂S); 3.90 ± 3.97 (m, CHN); 5.01 (br., NH);

7.22 ± 7.33 (m, 5 arom. H). ¹³C-NMR (100 MHz, CDCl₃): 22.2, 22.8 (Me); 24.5 (CH); 28.4 (Me); 30.7, 36.3, 43.3

(CH₂); 49.2, 49.3 (CH); 51.8 (Me); 79.2 (C); 127.1; 128.6, 128.9 (arom. C); 138.0, 155.6, 174.0 (C). FAB-MS: 396

(16, [M 1] ), 340 (100, [M À C₄H₈ 1] ), 296 (88, [M À Boc 1] ). Anal. calc. for C₂₁H₃₃NO₄S₂(395.56): C

63.77, H 8.41, N 3.54, S 8.11; found: C 63.81, H 8.33, N 3.59, S 8.21.

Methyl (1S)-2-{[(tert-butoxy)carbonyl]amino}propenoate (8). Compound 7b (25 mg, 0.06 mmol) was

dissolved in 3 ml of MeOH. NaIO₄(14 mg, 0.06 mmol) was dissolved in 0.5 ml of H₂O and added dropwise to the

first soln. at 08. The mixture was allowed to warm to r.t. within 1 h and stirred for an additional 2 h. Solvents were

removed, and the residue was extracted two times with CH₂Cl₂. The org. phase was washed with sat. NaHCO₃,

NH₄Cl, and NaCl solns., and dried (MgSO₄). Concentration under reduced pressure yielded a 1 :1 mixture of

the diastereoisomeric sulfoxides (NMR), which was subjected to thermal pyrolysis by refluxing in 5 ml of

toluene for 4 h (TLC). After removing toluene, FC (cyclohexane/AcOEt 6 :1) yielded pure 8 (11 mg, 63%).

Colorless solid. R_f0.46 (cyclohexane/AcOEt 6 :1). ¹H-NMR (400 MHz, CDCl₃): 0.91 (d, J 6.5, Me); 0.93

(d, J 6.0, Me); 1.42 (s, t-Bu); 1.47 ± 1.60 (m, CH₂, CH); 3.77 (s, MeO); 4.45 ± 4.51 (m, CHN); 5.11 (br., NH);

5.11 (br., 1 H, CCH₂); 6.17 (d, J 1.1, 1 H, CCH₂). ¹³C-NMR (100 MHz, CDCl₃): 22.3, 22.6 (Me); 25.1

(CH); 28.4 (Me); 43.9 (CH₂); 51.8 (Me); 79.3 (C); 126.0 (CH₂); 140.8, 155.1, 166.5 (C). FAB-MS: 307 (70, [M

Na] ), 272 (70, [M 1] ), 216 (100, [M À C₄H₈ 1] ), 172 (38, [M À Boc 1] ).

2-(Trimethylsilyl)ethyl (2S,3R)-4-(Benzylsulfanyl)-2-[(benzylsulfanyl)methyl]-3-{[(tert-butoxy)carbonyl]-

amino}butanoate (Boc-(2S,3R)-HCcy(Bn)₂-OTMSE; 9). Compound 7a (461 mg. 0.97 mmol) was dissolved in

10 mlof Me ₃SiCH₂CH₂OH; 460 mg (2 mmol) Ti(OC₂H₅)₄was added, and the soln. was stirred at 958 for 20 h.

After filtration over Celite, 40 mlof AcOEt was added, and the mixture was extracted with NaHCO ₃, NH₄Cl,

and NaClsonls. The org. phase was dried (MgSO ₄) and concentrated under reduced pressure. Residual

Me₃SiCH₂CH₂OH was removed by bulb-to-bulb distillation (1308/0.1 Torr). FC (cyclohexane/AcOEt 6 :1)

yielded 9 (420 mg, 77%). White solid. M.p. 53 ± 548. R_f0.7 (cyclohexane/AcOEt 7:1). [a]^r_D^:t: À4.8 (c 1.0,

CHCl₃). IR (KBr): 3402m, 2972m, 1725s, 1692s, 1507s, 1264m, 1247m, 1165s, 1040m, 947s, 862m, 836m, 696s.

¹H-NMR (400 MHz, CDCl₃): 0.04 (s, Me₃Si); 0.95 ± 1.00 (m, CH₂Si); 1.44 (s, t-Bu); 2.36 ± 2.42, 2.50 ± 2.56, 2.72 ±

2.78 (3m, 2 CH₂S); 3.00 ± 3.05 (m, CHCO); 3.73, 3.76 (2s, 2 PhCH₂S); 4.07 ± 4.18 (m, CHN, CH₂O); 5.39

(br., NH); 7.23 ± 7.36 (m, 10 arom. H). ¹³C-NMR (100 MHz, CDCl₃): À1.5 (Me); 17.5 (CH₂); 28.4 (Me); 30.8,

34.8, 36.0, 36.3 (CH₂); 46.5, 50.1 (CH); 63.5 (CH₂); 79.5 (C); 127.0, 127.1, 128.5, 128.6, 129.0, 129.1 (arom. C);

137.9, 138.0, 155.5, 173.3 (C). FAB-MS: 562 (22, [M 1] ), 506 (37), 470 (72, [M À Bn] ), 462 (32, [M À Boc

1] ), 434 (100), 414 (43), 386 (23). Anal. calc. for C₂₉H₄₃NO₄S₂Si (561.87): C 61.99, H 7.71, N 2.49, S 11.41;

found: C 61.89, H 7.59, N 2.51, S 11.41.

Boc-(2R,3R)-(a-CH₂OH)-b^2,3-HSer(OBn)-b³-HLeu-OMe (11). Compound

1 (0.93 g; 2.6 mmol) was

reacted with 1.0 mlof H ₂O₂(30%) and 175 mg of LiOH ´ H₂O (1.6 equiv.) in 12.5 mlof THF/H ₂O 4 :1 for

24 h at r.t. The mixture was treated with 1.26 g of Na₂SO₃in 5 mlof H ₂O and 1m HCl, and extracted with AcOEt/

CH₂Cl₂. The resulting compound was dissolved in 7 ml of H₂O and 10 mlof MeOH, and treated with 190 mg of

LiOH ´ H₂O (1.74 equiv.) for another 24 h. Acidification with 1m HClsonl . and extraction with CH ₂Cl₂gave

Helvetica Chimica Acta ± Vol. 83 (2000)

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0.92 g of the free Boc-b-amino acid. Boc-b³-HLeu-OMe (1.07 g, 3.9 mmol) was deprotected according to GP 1

and coupled according to GP 3a. Repeated recrystallization gave 11 (648 mg, 75%). Colorless solid. M.p. 118 ±

1198. R_f0.52 (Et₂O). [a]_D À18.5 (c 1.00, CHCl₃). IR (CHCl₃): 3426w, 3007m, 2957m, 1732m, 1702m, 1657m,

1499s, 1454w, 1368m, 1171s, 1089w, 1046w, 644w, 627w, 615w. ¹H-NMR (300 MHz, CDCl₃): 0.95 (d, J 6.5,

Me₂CH); 1.19 ± 1.28 (m, CH); 1.32 ± 1.45 (m, t-Bu, CH₂); 1.48 ± 1.64 (m, CH); v_A 2.36, v_B 2.44 (ABM, J_AB

15.6, J_AM 5.6, J_BM 5.3, CH₂C(O)); 2.70 ± 2.76 (m, CHC(O)); 3.44 ± 3.49 (m, CH₂O); 3.62 ± 3.67 (m, MeO, 1 H

of CH₂O); 3.72 ± 3.84 (m, 1 H of CH₂O); 4.11 ± 4.19 (m, CHN); 4.23 ± 4.31 (m, CHN); 4.51 (s, PhCH₂); 5.54

(d, J 9.7, BocNH); 6.41 (d, J 6.4, BocNH); 7.27 ± 7.38 (m, 5 arom. H). ¹³C-NMR (300 MHz, CDCl₃): 22.01;

22.85; 24.95; 28.31; 39.09; 43.09; 44.30; 48.52; 49.24; 51.78; 61.53; 70.69; 73.19; 76.94; 127.76; 127.89; 128.50;

137.72; 156.59; 171.68; 172.17. FAB-MS: 983.4 (9.9, [2M Na] ), 503.2 (57.7, [M Na] ), 481.3 (76.5, [M

H] ), 381.2 (100.0, [M À t-Bu 2 H] ).

Boc-b³-HVal-(2R,3R)-(a-CH₂OH)-b^2,3-HSer(OBn)-b³-HLeu-OMe (12a). Compound 11 (520 mg, 1.38 mmol)

was deprotected according to GP 1 and coupled to Boc-b³-HVal-OH (240 mg, 1.05 mmol) according to GP 6.

FC (CH₂Cl₂/MeOH 90 :8) gave 12a (178 mg, 26%). Colorless solid. [a]_D À25.7 (c 0.66, CHCl₃). IR

(CHCl₃): 3420m, 3003m, 2962s, 1704s, 1657s, 1499s, 1454w, 1391m, 1367m, 1173s, 1096w, 1044w, 1014w, 913w,

861w. ¹H-NMR (300 MHz, (D₆)DMSO, 508): 0.75 ± 0.83 (m, 12 H); 1.10 ± 1.19 (m, 1 H); 1.34 (s, 10 H); 1.61 ±

1.68 (m, 2 H); 2.17 ± 2.19 (m, 2 H); 2.34 ± 2.36 (m, 2 H); 2.57 ± 2.61 (m, 1 H); 3.27 ± 3.39 (m, 2 H); 3.45 ± 3.62

(m, 3 H); 3.53 (s, 3 H); 3.87 ± 3.89 (m, 2 H); 4.14 ± 4.36 (m, 2 H); 4.37 ± 4.47 (m, 3 H); 7.26 ± 7.33 (m, 5 H).

Boc-(R)-b³-HSer(Bn)-(S)-b³-HLeu-OMe (14). Boc-(S)-b³-HLeu-OMe (560 mg, 2.16 mmol) was depro-

tected according to GP 1, dissolved in 4 ml of CHCl₃, and treated with Et₃N (1.09 g, 10.8 mmol), HOBt (350 mg,

2.6 mmol), Boc-(R)-b³-HSer(Bn)-OH (668 mg, 2.16 mmol) in 4 ml of CHCl₃, and EDC (480 mg, 2.6 mmol)

according to GP 3a. FC (CH₂Cl₂/MeOH 20 :1) yielded 14 (878 mg, 90%). White solid. M.p. 102 ± 1038. R_f0.6

(CH₂Cl₂/MeOH 20 :1). [a]^r_D^:t: À11.0 (c 1.0, CHCl₃). IR (KBr): 3269s, 3068m, 2961s, 2870m, 1738s, 1696s,

1653s, 1547s, 1451m, 1364m, 1294m, 1252m, 1179m, 1085m, 1066m, 862w, 746m, 698m. ¹H-NMR (400 MHz,

CDCl₃): 0.89 (d, J 6.6, Me); 0.90 (d, J 6.5, Me); 1.23 ± 1.30 (m, 1 H, CH₂); 1.40 ± 1.46 (m, t-Bu, 1 H of CH₂);

1.52 ± 1.62 (m, CH); 2.41 ± 2.54 (m, 2 CH₂CO); 3.48 (dd, J 9.4, 6.2, 1 H, CH₂O); 3.60 (br., 1 H, CH₂O); 3.66

(s, MeO); 4.04 ± 4.12 (m, CHN); 4.27 ± 4.36 (m, CHN); 4.51 (s, PhCH₂O); 5.45 (br., NH); 6.20 (br., NH); 7.27 ±

7.37 (m, 5 arom. H). ¹³C-NMR (100 MHz, CDCl₃): 22.1, 22.9 (Me); 25.0 (CH); 28.4 (Me); 38.1, 38.9, 43.1 (CH₂);

44.2 (CH); 51.7 (Me); 71.2, 73.2 (CH₂); 79.4 (C); 127.7; 127.8, 128.5 (arom. C); 138.0, 155.5, 170.1, 172.2 (C).

FAB-MS: 473 (14, [M Na] ), 451 (48, [M 1] ), 351 (100, [M À Boc 1] ). Anal. calc. for C₂₄H₃₈N₂O₆

(450.57): C 63.98, H 8.50, N 6.22; found: C 64.01, H 8.43, N 6.25.

Boc-(R)-b³-HVal-(R)-b³-HSer(Bn)-(S)-b³-HLeu-OMe (16a). Compound 14 (849 mg, 1.88 mmol) was

deprotected according to GP 1, dissolved in CHCl₃(3.5 ml), and treated with Et₃N (960 mg, 9.5 mmol), HOBt

(311 mg, 2.3 mmol), Boc-(R)-b³-HVal-OH (436 mg, 1.88 mmol) in CHCl₃(3.5 ml), and EDC (440 m, 2.3 mmol)

according to GP 3a. FC (CH₂Cl₂/MeOH 20 :1) yielded 16a (987 mg, 92%). White solid. M.p. 142 ± 1438. R_f0.65

(CH₂Cl₂/MeOH 20 :1). [a]^r_D^:t: À12.7 (c 1.0, CHCl₃). IR (KBr): 3308s, 2960s, 2871m, 1741m, 1687s, 1647s,

1541s, 1438w, 1367m, 1310w, 1248w, 1174m, 1044w, 1022w, 867w, 735m, 697m. ¹H-NMR (400 MHz, CDCl₃):

0.89 ± 0.92 (m, 4 Me); 1.22 ± 1.29 (m, 1 H, CH₂); 1.39 ± 1.49 (m, t-Bu, 1 H of CH₂); 1.51 ± 1.62 (m, CH); 1.76 ± 1.84

(m, CH); 2.26 ± 2.54 (m, 3 CH₂CO); 3.48 (dd, J 9.5, 6.4, 1 H, CH₂O); 3.62 ± 3.72 (m, MeO, 1 H of CH₂O,

CHN); 4.25 ± 4.37 (m, 2 CHN); 4.51 (s, PhCH₂O); 5.16 (br., NH); 6.50 (br., NH); 6.80 (br., NH); 7.27 ± 7.37

(m, 5 arom. H). ¹³C-NMR (100 MHz, CDCl₃): 18.5, 19.5, 22.1, 22.2 (Me); 25.0 (CH); 28.4 (Me); 32.2 (CH);

37.9; 39.2, 43.2 (CH₂); 44.4, 46.9 (CH); 51.7 (Me); 53.7 (CH); 70.8,73.3 (CH₂); 79.1 (C), 127.8, 127.9, 128.5

(arom. C); 138.0, 156.0, 170.3, 171.0, 172.3 (C). FAB-MS: 586 (100, [M Na] ), 564 (72, [M 1] ), 464 (79,

[M À Boc 1]⁾. Anal. calc. for C₃₀H₄₉N₃O₇(563.73): C 63.92, H 8.76, N 7.45; found: C 63.91, H 8.55, N 7.44.

Boc-(R)-b³-HVal-(R)-b³-HSer(Bn)-(S)-b³-HLeu-OH (16b). Compound 16a (395 mg, 0.7 mmol) was

dissolved in 3 ml of MeOH/CF₃CH₂OH 1:1 and treated with 2 mlof 5 n NaOH according to GP 2b (reaction

time 14 h) to yield 16b (378 mg, 98%). White solid. M.p. 188 ± 1898. R_f0.45 (CH₂Cl₂/MeOH 15 :1). [a]^r_D^:t: 1.7

(c 0.5, MeOH). IR (KBr): 3332w, 2960s, 2871m, 2475m, 1721s, 1679s, 1630s, 1590m, 1528w, 1456s, 1367m,

1310w, 1250w, 1175m, 1122m, 1020w, 968w, 865w, 776w, 738m, 697m. ¹H-NMR (400 MHz, CD₃OD): 0.86 ± 0.93

(m, 4 Me); 1.26 ± 1.33 (m, 1 H, CH₂); 1.41 ± 1.49 (m, t-Bu, 1 H of CH₂); 1.55 ± 1.65 (m, CH); 1.69 ± 1.77 (m, CH);

2.20 ± 2.50 (m, 3 CH₂CO); 3.46 ± 3.56 (m, CH₂O); 3.71 ± 3.76 (m, CHN); 4.25 ± 4.32 (m, CHN); 4.33 ± 4.39

(m, CHN); 4.51 (s, PhCH₂O); 7.23 ± 7.34 (m, 5 arom. H). ¹³C-NMR (100 MHz, CD₃OD): 18.3, 19.7, 22.2, 23.7

(Me); 26.1 (CH); 28.9 (Me); 33.6 (CH); 39.1, 40.2, 41.0, 44.5 (CH₂); 45.9, 48.4 (CH); 54.9 (CH); 72.0, 74.3

(CH₂); 80.0 (C), 128.7, 128.9, 129.4 (arom. C); 139.6, 158.0, 172.3, 173.5, 174.9 (C). FAB-MS: 572 (100, [M

Na] ), 550 (21, [M 1] ), 450 (33, [M À Boc 1] ).

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Boc-(b³-HVal-(2R,3R)-(a-CH₂OH)-b^2,3-HSer(OBn)-b³-HLeu)₂-OMe (17). Compound 12a (177 mg,

0.298 mmol) was dissovl ed in 5.5 mlof MeOH, 0.6 mlof 1 m NaOH was added, and the resulting mixture was

stirred at r.t. for 2 d. The pH of the resulting soln. was adjusted to 2 using 1m HClsoln., and the precipitate was

collected by filtration resulting in 12b (92 mg, 52%). The other fragment was obtained by deprotecting 12a

according to GP 1. Both fragments were coupled according to GP 3a. FC (CH₂Cl₂/MeOH 10 :1) gave 17

(108.6 mg, 66%). Colorless solid. CD: 220 (À25000), 200 (125000). ¹H-NMR (300 MHz, CD₃OD): 0.88 ± 0.94

(m, 8 Me); 1.21 ± 1.50 (m, 4 H); 1.43 (s, t-Bu); 1.60 ± 1.82 (m, 4 H); 2.26 ± 2.53 (m, 8 HÀC(a)); 2.70 ± 2.76

(m, 1 H); 2.90 ± 2.94 (m,1 H); 3.43 ± 3.51 (m,2 CH₂O); 3.62 ± 3.70 (m, 2 CH₂O, MeO); 3.79 ± 3.86 (m, HÀC(b));

4.35 ± 4.51 (m, 2 PhCH₂, 4 HÀC(b)). FAB-MS: 1208.4 (10, [M {3-NOBA}] ), 1077.5 (100, [M Na] ), 955.5

(8, [M À Boc] ).

H₂N-(b³-HVal-(2R,3R)-(a-CH₂OH)-b^2,3-HSer(OBn)-b³-HLeu)₂-OH (18). b-Peptide 17 (55.5 mg,

0.525 mmol) was dissolved in 2 ml of TFE. NaOH (210 mg, 100 equiv.) dissolved in 1 ml of H₂O was added,

and the resulting suspension was stirred overnight at r.t. The resulting clear soln. was acidified with 6m HClto

pH 2 and extracted with AcOEt. The org. phase was dried (MgSO₄) and evaporated. The resulting 54.5 mg

(0.523 mmol) Boc-b-peptide acid were deprotected according to GP 1. The resulting colorless oil was dissolved

in 5 mlof THF, 0.5 mlof AcOH, and 10 mg of Pd/C (10%) were added, and the apparatus was evacuated and

flushed with H₂three times. The mixture was stirred for 40 h at r.t. The resulting suspension was filtered over

Celite and evaporated. Purification by prep. RP-HPLC gave 18 (9.2 mg; 23%) after lyophilization. Colorless

fluffy solid. Anal. RP-HPLC: t_R15.3 min (C₁₈; gradient 20 min 25 ± 50% B; 10 min 50 ± 99% B). ¹H-NMR

(500 MHz, CD₃OH): chemicalshifts of non-Me protons of 18 were assigned by 2D-NMR (DQF-COSY,

TOCSY). Xaa (2R,3R)-(a-CH₂OH)-b^2,3-HSer(OBn).

Val1

Xaa2

Leu3

Val4

Xaa5

Leu6

NH

n/d

3.39

1.91

±

2.68

2.53

±

7.95

4.11

3.57

3.48

2.71

±

7.98

4.15

1.34

1.18

2.38

2.31

±

7.91

3.99

1.69

±

2.49

2.26

±

7.71

4.08

3.51

3.44

2.64

±

7.89

4.21

1.37

1.24

2.47

2.36

±

HÀC(b)

HÀC(g)

H'ÀC(g)

HÀC(a)

H'ÀC(a)

HÀC(b')

H'ÀC(b')

3.65

3.61

±

FAB-MS: 799.6 (67, [M K] ), 783.6 (100, [M Na] ), 761.6 (67, [M H] ).

Boc-(2S,3R)-HCcy(Bn)₂-(R)-b³-HVal-(S)-b³-HAla-(S)-b³-HLeu-OMe (19). Compound

9

(200 mg,

0.36 mmol) was dissolved in 4 ml of THF and cooled to 08. TBAF (220 mg, 0.7 mmol) was added, and the

soln. was stirred at r.t. for 1 h. H₂O (5 ml) and Et₂O (5 ml) were added, and the mixture was stirred for 10 min.

The phases were divided, and the aq. phase was extracted with AcOEt. The org. phases were dried (MgSO₄) and

then concentrated under reduced pressure: 10 was obtained as a colorless oil and used without further

purification. In a second flask, 15a (165 mg, 0.36 mmol) was deprotected according to GP 1, dissolved in 1 ml of

CHCl₃, and treated with NMM (145 mg, 1.44 mmol), HOBt (65 mg, 0.48 mmol), 10 (see above) in 1 mlof

CHCl₃, and EDC (92 mg, 0.48 mmol) according to GP 3a, FC (CH₂Cl₂/MeOH 12 :1) yielded 19 (180 mg, 63%).

White solid. M.p. 169 ± 1708. R_f0.5 (CH₂Cl₂/MeOH 15 :1). [a]^r_D^:t: À7.3 (c 1.0, CHCl₃): IR (KBr): 3301m,

2963m, 1735m, 1718m, 1686s, 1654s, 1560s, 1542s, 1262m, 1247m, 1170m, 802m, 696s. ¹H-NMR (500 MHz,

CDCl₃): 0.90 ± 0.92 (m, 4 Me); 1.18 (d, J 6.7, Me); 1.25 ± 1.32 (m, 1 H, CH₂); 1.42 (s, t-Bu); 1.43 ± 1.50 (m, 1 H,

CH₂); 1.54 ± 1.62 (m, CH); 1.79 ± 1.86 (m, CH); 2.18 ± 2.82 (m, 3 CH₂CO, CHCO, 2 CH₂S); 3.67 (s, MeO); 3.72,

3.75 (2s, 2 PhCH₂); 3.91 ± 3.97 (m, CHN); 4.00 ± 4.06 (m, CHN); 4.10 ± 4.18 (m, CHN); 4.27 ± 4.36 (m, CHN);

6.08 (br., NH); 6.27 (br., NH); 6.53 (br., NH); 6.94 (br., NH); 7.21 ± 7.35 (m, 10 arom. H). ¹³C-NMR (125 MHz,

CDCl₃): 18.9, 19.5, 19.6, 22.2, 22.8 (Me); 25.1 (CH); 28.4 (Me); 31.6 (CH); 34.8, 36.0, 36.9, 38.4, 40.0, 41.3 (CH₂);

43.2, 44.4, 46.7, 50.9 (CH); 51.8 (Me); 52.2 (CH); 79.1 (C); 127.0, 127.2, 128.5, 128.6, 128.9, 129.1 (arom. C);

138.2, 138.3, 155.7, 170.0, 170.7, 172.0, 172.3 (C). FAB-MS: 823 (5, [M Na] ), 801 (25, [M 1] ), 701 (100,

[M À Boc 1] ). Anal. calc. for C₄₂H₆₄N₄O₇S₂(801.11): C 62.97, H 8.05, N 6.99; found: C 62.63, H 7.73, N 6.92.

Boc-(2S,3R)-HCcy(Bn)₂-(R)-b³-HVal-(R)-b³-HSer(Bn)-(S)-b³-HLeu-OMe (20). Compound 9 (216 mg,

0.385 mmol) was dissolved in 4 ml of THFand cooled to 08. TBAF (240 mg, 0.76 mmol) was added, and the soln.

was stirred at r.t. for 1 h. H₂O (5 ml) and Et₂O (5 ml) were added, and the mixture was stirred for 10 min. The

Helvetica Chimica Acta ± Vol. 83 (2000)

2137

phases were divided, and the aq. phase was extracted with AcOEt. The org. phases were dried (MgSO₄) and

then concentrated under reduced pressure: 10 was obtained as a colorless oil and used without further

purification. In a second flask, 16a (217 mg, 0.385 mmol) was deprotected according to GP 1, dissolved in 1 ml of

CHCl₃, and treated with NMM (155 mg, 1.54 mmol), HOBt (68 mg, 0.5 mmol). 10 (see above) in 1 mlof CHCl ₃,

and EDC (96 mg, 0.5 mmol) according to GP 3a. FC (CH₂Cl₂/MeOH 20 :1) yielded 20 (110 mg, 34%). White

solid. M.p. 146 ± 1478. R_f0.55 (CH₂Cl₂/MeOH 15 :1). [a]^r_D^:t: À2.3 (c 0.5, CHCl₃). IR (KBr): 3297s, 3063w,

3030w, 2958m, 1740m, 1690s, 1647s, 1541s, 1496m, 1452m, 1437m, 1367m, 1290w, 1248w, 1170m, 1028w, 699w.

¹H-NMR (400 MHz, CDCl₃): 0.88 ± 0.95 (m, 4 Me); 1.20 ± 1.27 (m, 1 H, CH₂); 1.35 ± 1.48 (m, t-Bu, 1 H of CH₂);

1.51 ± 1.61 (m, CH); 1.79 ± 1.85 (m, CH); 2.25 ± 2.82 (m, 3 CH₂CO, CHCO, 2 CH₂S); 3.65 (s, MeO); 3.71, 3.74

(2s, 2 PhCH₂S); 3.90 ± 4.05 (m,2 CHN); 4.23 ± 4.36 (m, 2 CHN); 4.47 (s, PhCH₂O); 6.13 (br., NH); 6.39

(br., NH); 6.53 (br., NH); 6.90 (br., NH); 7.19 ± 7.36 (m, 10 arom. H). ¹³C-NMR (100 MHz, CDCl₃): 18.9, 19.5,

22.1, 22.8 (Me); 25.0 (CH); 28.4 (Me); 31.4 (CH); 31.8, 34.8, 35.9, 36.9, 37.2, 38.2, 38.9, 43.2 (CH₂); 44.4, 46.6,

50.8 (CH); 51.7 (Me); 52.1 (CH); 79.1 (C); 127.0, 127.2, 127.8, 127.9, 128.5, 128.6, 128.9, 129.1 (arom. C); 137.8,

138.2, 138.3, 155.7, 170.4, 172.0, 172.3 (C). FAB-MS: 929 (100, [M Na] ), 907 (39, [M 1] ), 807 (95, [M À

Boc 1] ).

Boc(R)-b³-HVal-(S)-b³-HAla-(S)-b³-HLeu-(2S,3R)-HCcy(Bn)₂-(R)-b³-HVal-(S)-b³-HAla-(S)-b³-HLeu-

OMe (21). Compound 19 (140 mg, 0.175 mmol) was deprotected according to GP 1, dissolved in 0.5 ml of CHCl₃,

and treated with NMM (71 mg, 0.7 mmol), HOBt (54 mg, 0.4 mmol), 15b (78 mg, 0.175 mmol) in 0.6 ml of

DMF, and EDC (76 mg, 0.4 mmol) according to GP 3b. Recrystallization from MeOH yielded 21 (156 mg,

79%). White solid. M.p. 243 ± 2448. R_f0.33 (CH₂Cl₂/MeOH 10 :1). [a]^r_D^:t: À7.3 (c 1.0, CHCl₃). IR (KBr):

3304s, 2960m, 1741m, 1684s, 1636s, 1541s, 1457s, 1261m, 1175m, 1142m, 1018m, 804w, 698m. ¹H-NMR (500 MHz,

CDCl₃/CD₃OD 3 :1): 0.88 ± 0.96 (m, 6 Me); 1.13, 1.16 (2d, J 6.7, 2 Me); 1.27 ± 1.35 (m, 3 CH); 1.43 (s, t-Bu);

1.43 ± 1.48 (m, 1 H, CH₂); 1.57 ± 1.64 (m, 2 H, 2 CH₂); 1.74 ± 1.85 (m, 2 CH); 2.21 ± 2.78 (m, 7 CH₂CO, 2 CH₂S);

3.67 ± 3.73 (m, CHN); 3.68 (s, MeO); 3.71, 3.74 (2s, 2 PhCH₂S); 4.10 ± 4.17 (m, 2 CHN); 4.22 ± 4.32 (m, 3 CHN);

4.33 ± 4.37 (m, CHN); 5.87 (br., NH); 7.21 ± 7.33 (m, 10 arom. H); 7.41 (br., NH); 7.53 (br., NH). ¹³C-NMR

(125 MHz, CDCl₃/CD₃OD 3 :1): 18.1, 18.5, 18.9, 19.0, 19.7, 21.6, 21.7, 22.5, 22.5, 24.6 (Me); 24.6 (CH); 27.9 (Me);

31.3 (CH₂); 31.6, 32.0 (CH); 33.9, 35.5, 36.0, 37.3, 38.3, 39.3, 39.7, 41.0, 41.3, 41.9 (CH₂); 42.7 (CH); 42.7, 43.2

(CH₂); 44.2, 44.6, 46.5, 49.0, 51.4 (CH); 51.7 (Me); 53.2 (CH); 78.8 (C); 126.7, 126.9, 128.2, 128.3, 128.5, 128.7,

130.0 (arom. C); 137.6, 137.9, 156.2, 170.3, 170.5, 170.8, 171.3, 172.0, 172.0 (C). FAB-MS: 1149 (100, [M Na] ),

1126 (5, [M 1] ), 1026 (15, [M À Boc 1] ).

Boc-(R)-b³-HVal-(R)-b³-HSer(Bn)-(S)-b³-HLeu-(2S,3R)-HCcy(Bn)₂-(R)-b³-HVal-(R)-b³-HSer(Bn)-

(S)-b³-HLeu-OMe (22). Compound 20 (98 mg, 0.11 mmol) was deprotected according to GP 1, dissolved in

0.5 mlof CHCl ₃and treated with Et₃N (61 mg, 0.6 mmol), HOBt (27 mg, 0.2 mmol), 16b (61 mg, 0.11 mmol) in

0.3 mlof DMF, and EDC (38 mg, 0.2 mmol) according to GP 3b. FC (CH₂Cl₂/MeOH 20 :1) yielded 22 (117 mg,

80%). White solid. CD (0.2 mm in MeOH): 6.2 ´ 10⁴(199 nm); À2.7 ´ 10⁴(215 nm). IR (KBr): 3297s, 3065w,

2958m, 2870m, 1734m, 1685s, 1653s, 1541s, 1452w, 1366m, 1311w, 1248m, 1175m, 1122w, 1028w, 735w, 698w.

¹H-NMR (400 MHz, CDCl₃/CD₃OD 3 :1): 0.77 ± 0.87 (m, 8 Me); 1.13 ± 1.35 (m, 4 H of 4 CH₂, t-Bu); 1.50 ± 1.58

(m, 2 CH); 1.60 ± 1.69 (m, 2 CH); 2.13 ± 2.62 (m, 6 CH₂CO, CHCO, 2 CH₂S); 3.31 ± 3.69 (m, MeO, 2 PhCH₂S,

3 CHN); 3.96 ± 4.35 (m, 3 CHN); 4.41 (s, PhCH₂O); 4.51 (s, PhCH₂O); 6.03 (br., NH); 7.15 ± 7.26 (m, 20 ar-

om. H). FAB-MS: 1377 (14, [M K] ), 1361 (100, [M Na] ), 1339 (6, [M 1] ), 1239 (79, [M À Boc 1] ).

H-(R)-b³-HVal-(S)-b³-HAla-(S)-b³-HLeu-(2S,3R)-ADTC-(R)-b³-HVal-(S)-b³-HAla-(S)-b³-HLeu-OH ´

CF₃CO₂H (23). Compound 21 (83 mg, 0.074 mmol) was dissolved in 2 ml of CF ₃CH₂OH and treated with 2 ml

of 5n NaOH at 458 over 40 h. The mixture was diluted with H₂O, and the pH was adjusted to 2 ± 3 with 5n HCl

(08). The soln. was extracted with AcOEt, and the combined org. phases washed successively with sat. NaCl

soln. and H₂O. The org. phase was evaporated and dried under h.v. The resulting white solid (80 mg) was

deprotected and cyclized according to GP 4. The crude peptide was purified by prep. RP-HPLC (30 ! 70% B in

35 min) according to GP 5 to yield 23 (22 mg, 36%). White solid. RP-HPLC (45 ! 60% B in 20 min): t_R24.1 min,

purity >98%. M.p. 149 ± 1518 (dec.). CD (0.2 mm in MeOH): À1.03 ´ 10⁵(209 nm); 1.4 ´ 10⁴(289 nm). CD

(0.2 mm in H₂O): À9.7 ´ 10⁴(205 nm); 1.1 ´ 10⁴(281 nm). IR (KBr): 3292s, 3082m, 2964m, 1734m, 1654s, 1542s,

1458m, 1374m, 1261m, 1202m, 1137m, 800w, 699w. ¹H-NMR (500 MHz, CD₃OD): 0.84 ± 0.93 (m, 6 Me); 1.05 ±

1.10 (m, 3 Me); 1.18 (d, J 6.7, Me); 1.21 ± 1.39 (m, 4 CH); 1.50 ± 1.61 (m, CH₂); 1.62 ± 1.72 (m, 1 H, CH₂); 2.01 ±

3.12 (m, 1 H of CH₂, 6 CH₂CO, CHCO, 2 CH₂S); 3.50 ± 3.54 (m, CHN); 4.10 ± 4.18 (m, CHN); 4.23 ± 4.29

(m, CHN); 4.35 ± 4.43 (m, CHN); 4.44 ± 4.48 (m, CHN); 4.49 ± 4.56 (m, 2 CHN); 7.41 (br., NH); 8.39 (br., NH);

8.57 (br., NH). ¹³C-NMR (125 MHz, CD₃OD): 18.3, 19.2, 19.5, 19.9, 21.0, 21.5, 23.0, 23.1, 23.4, 23.5 (Me); 25.9,

26.1, 32.0, 34.3 (CH); 35.9, 37.9, 39.4, 40.2, 40.4, 42.3, 42.9, 43.3 (CH₂); 43.5, 45.3, 45.6 (CH); 46.0, 46.9 (CH₂);

50.3, 51.8, 53.2, 56.0 (CH); 171.2, 171.4, 171.6, 172.0, 173.1, 173.2, 175.2 (C). FAB-MS: 1660 (4, [2M 1] ), 852

2138

Helvetica Chimica Acta ± Vol. 83 (2000)

(4, [M Na] ), 830 (100, [M 1] ). ESI-MS (pos.): 869 (6, [M K] ), 853 (36, [M Na] ), 831 (100, [M

1] ). ESI-MS (neg.): 828 (100, [M À 1]^À).

H-(R)-b³-HVal-(R)-b³-HSer-(S)-b³-HLeu-(2S,3R)-ADTC-(R)-b³-HVal-(R)-b³-HSer-(S)-b³-HLeuÀOH ´

CF₃CO₂H (24). Compound 22 (108 mg, 0.08 mmol) was dissolved in 2 ml of CF ₃CH₂OH and treated with 2 ml

of 5n NaOH at 458 over 17 h. The mixture was diluted with H₂O, and the pH was adjusted to 2 ± 3 with 5n HCl

(08). The soln. was extracted with AcOEt, and the combined org. phases were washed successively with sat.

NaClsoln. and H ₂O. The org. phase was evaporated and dried under h.v. The resulting white solid (106 mg) was

deprotected and cyclized according to GP 4. The crude peptide was purified by prep. RP-HPLC (20 ! 50% B in

45 min) according to GP 5 to yield 24 (23 mg, 36%). White solid. RP-HPLC (30 ! 45%, B in 30 min):

t_R26.1 min, purity >98%. M.p. 145 ± 1468 (dec.). CD (0.2 mm in MeOH): À1.08 ´ 10⁵(208 nm); 1.2 ´ 10⁴

(285 nm). CD (0.2 mm in H₂O): À1.00 ´ 10⁵(206 nm); 0.9 ´ 10⁴(286 nm). IR (KBr): 3293s, 3092w, 2963m,

1654m, 1550s, 1466w, 1430w, 1388w, 1203s, 1137s, 1057w, 1028w, 801w, 723m. ¹H-NMR (500 MHz, CD₃OD):

0.90 ± 0.99 (m, 6 Me); 1.10 ± 1.13 (m, 2 Me); 1.24 ± 1.73 (m, 3 CH, 2 CH₂); 2.06 ± 3.17 (m, 1 H of CH₂, 6 CH₂CO,

CHCO, 2 CH₂S); 3.44 ± 3.62 (m, 2 CH₂O, CHN), 4.21 ± 4.26 (m, CHN); 4.30 ± 4.36 (m, CHN); 4.38 ± 4.50

(m, 2 CHN); 4.51 ± 4.60 (m, 2 CHN); 7.41 (br., NH); 7.68 (br., NH); 7.84 (br., NH); 8.28 (br., NH); 8.43

(br., NH); 8.62 (br., NH). ¹H-NMR (500 MHz, H₂O/D₂O 9 :1): 0.83 ± 0.90 (m, 6 Me); 0.99 ± 1.03 (m, 2 Me);

1.23 ± 1.76 (m, 3 CH, 2 CH₂); 1.96 ± 2.94 (m, 1 H of CH₂, 6 CH₂CO, CHCO, CH₂S); 3.10 ± 3.14 (m, CH₂S); 3.51 ±

3.63 (m, 2 CH₂O, CHN); 4.04 ± 4.08 (m, CHN); 4.06 ± 4.10 (m, CHN); 4.17 ± 4.21 (m, CHN); 4.18 ± 4.23

(m, CHN); 4.23 ± 4.28 (m, CHN); 4.25 ± 4.30 (m, CHN); 7.87 (br., NH); 7.89 (br., NH); 7.91 (br., NH); 7.96

(br., NH); 8.17 (br., NH); 8.27 (br., NH). ¹³C-NMR (125 MHz, H₂O/D₂O 9 :1): 19.9, 20.2, 20.3, 21.0, 24.2, 24.2,

25.0, 25.0 (Me); 27.1, 27.2, 32.9, 34.8 (CH); 37.0, 39.0, 39.4, 39.5, 39.8, 39.9, 40.4, 40.5, 40.7, 43.0, 44.2, 46.0, 46.8

(CH₂); 47.9, 48.1, 51.2, 51.3, 51.8, 52.9 (CH); 53.5 (CH₂); 54.7, 57.0, 57.1 (CH); 65.7, 66.1 (CH₂); 174.3, 174.6, 174.7,

174.9, 175.0, 175.3, 179.0 (C). FAB-MS: 884 (51, [M Na] ), 862 (100, [M 1] ).

Boc-trans-ACHC-(R)-b³-HVal-(S)-b³-HAla-(S)-b³-HLeu-OMe (25a/b). Compound 15a (458 mg, 1 mmol)

was deprotected according to GP 1, dissolved in 1.8 ml of CHCl₃, and treated with Et₃N (505 mg, 5 mmol),

HOBt (165 mg, 1.2 mmol), Boc-trans-ACHCÀOH (243 mg, 1 mmol) in 1.8 ml of CHCl₃, and EDC (230 mg,

1.2 mmol) according to GP 3a. Recrystallization from MeOH yielded 25a/25b (411 mg, 72%). White solid. M.p.

169 ± 1708. R_f0.4 (CH₂Cl₂/MeOH 15 :1). IR (KBr): 3304s, 3297s, 3080w, 2959m, 2931m, 2871w, 1744m, 1684s,

1646s, 1541s, 1458w, 1438w, 1368m, 1320w, 1270w, 1174m, 1141w, 1051w, 1001w. ¹H-NMR (300 MHz, CDCl₃/

CD₃OD 3 :1; 2 epimers): 0.81 ± 0.85 (m, 3 Me); 1.06, 1.08 (2d, J 6.2, Me); 1.32 (s, t-Bu); 1.17 ± 2.43 (m, 2 CH,

5 CH₂, 3 CH₂CO, CHCO); 3.42 ± 3.46 (m, CHN); 3.59 (s, MeO); 3.85 ± 3.93 (m, CHN); 3.95 ± 4.6 (m, CHN);

4.10 ± 4.18 (m, CHN). FAB-MS: 605 (42, [M Na] ), 583 (45, [M 1] ), 483 (17, [M À Boc 1] ).

Boc-(R)-b³-HVal-(S)-b³-HAla-(S)-b³-HLeu-trans-ACHC-(R)-b³-HVal-(S)-b³-HAla-(S)-b³-HLeu-OMe

(26a/26b). The epimer mixture 25a/25b (291 mg, 0.5 mmol) was deprotected according to GP 1, dissolved in 3 ml

of DMF/CHCl₃1 :1 and treated with Et₃N (253 mg, 2.5 mmol), HOBt (81 mg, 0.6 mmol), 15b (243 mg, 1 mmol)

in 2 mlof DMF, and EDC (115 mg, 0.6 mmol) according to GP 3b. The resulting crude 26a/26b (444 mg) could

not be purified due to low solubility in all common org. solvents. White solid. M.p. 201 ± 2028. R_f0.4 (CH₂Cl₂/

MeOH 10 :1): IR (KBr): 3296s, 3081w, 2959m, 2933m, 2872w, 1740m, 1689s, 1649s, 1543s, 1438m, 1367m, 1311m,

1249m, 1175m, 1144w, 1052w, 1020w. ¹H-NMR (500 MHz, CDCl₃/CD₃OD 3 :1; 2 epimers): 0.81 ± 0.87

(m, 8 Me); 1.02 ± 1.30 (m, 2 Me, 3 CH₂); 1.35, 1.38 (2s, t-Bu); 1.47 ± 2.21 (m, 3 CH₂, 4 CH); 2.25 ± 3.15

(m, 6 CH₂CO, CHCO); 3.60, 3.65 (2s, MeO); 3.62 ± 4.36 (m, 7 CHN). FAB-MS: 1149 (100, [M Na] ), 1126

(5, [M 1] ), 1026 (15, [M À Boc 1] ).

H-(R)-b³-HVal-(S)-b³-HAla-(S)-b³-HLeu-(2R,3R)-ACHC-(R)-b³-HVal-(S)-b³-HAla-(S)-b³-HLeu-OH ´

CF₃CO₂H (27a) and H-(R)-b³-HVal-(S)-b³-HAla-(S)-b³-HLeu-(2S,3S)-ACHC-(R)-b³-HVal-(S)-b³-HAla-(S)-

b³-HLeu-OH ´ CF₃CO₂H (27b). The epimer mixture 26a/26b (59 mg, 0.052 mmol) was deprotected according to

GP 1 and GP 2b. The crude peptide was purified by prep. RP-HPLC (10 ! 40% B in 35 min) according to GP 5

to yield 27a (13 mg, 31%) and prep. RP-HPLC (20 ! 60% B in 45 min) to yield 27b (11 mg, 26%).

Data for 27a: White solid. RP-HPLC (0 ! 40% B in 45 min): t_R32.9 min, purity >99%. M.p. 2808 (dec.).

CD (0.2 mm in MeOH): À3.2 ´ 10⁴(200 nm); 0.8 ´ 10⁴(214 nm); 0.5 ´ 10⁴(228 nm). IR (KBr): 3293s, 3081w,

1

2960m, 2935m, 1654s, 1543s, 1458w, 1388w, 1312w, 1202m, 1140m, 800w, 722w. H-NMR (500 MHz, CD₃OD):

0.89 ± 0.93 (m, 6 Me); 1.06 (dd, J 6.9, 3.9, 2 Me); 1.13 (d, J 6.7, Me); 1.19 (d, J 6.3, Me); 1.22 ± 2.04

(m, 4 CH, 6 CH₂); 2.21 ± 2.62 (m, 6 CH₂CO, CHCO); 3.40 ± 3.45 (m, CHN); 3.90 ± 3.97 (m, CHN); 4.03 ± 4.08

(m, CHN); 4.11 ± 4.23 (m, 2 CHN); 4.25 ± 4.34 (m, 2 CHN). ¹³C-NMR (125 MHz, CD₃OD): 18.3, 18.7, 18.8,

19.9, 20.2, 20.9, 22.3, 22.5, 23.5, 23.7 (Me); 26.0 (CH); 26.0 (CH₂); 26.1 (CH); 26.3, 31.3 (CH₂); 31.9, 33.4 (CH);

33.8, 35.7, 39.8, 41.0, 41.7, 43.4, 44.4 (CH₂); 44.5, 44.6 (CH); 44.8 (CH₂); 45.9, 46.3, 50.7, 52.2, 53.0, 55.9 (CH);

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2139

171.7, 172.2, 172.5, 172.6, 172.7, 174.9, 175.9 (C). FAB-MS: 832 (10, [M K] ), 816 (100, [M Na] ), 794 (81,

[M 1] ).

Data for 27b: White solid. RP-HPLC (20 ! 55% B in 20 min): t_R19.0 min, purity >98%. M.p. 148 ± 1498

(dec.). CD (0.2 mm in MeOH): 8.9 ´ 10⁴(198 nm); À6.2 ´ 10⁴(216 nm). CD (0.2 mm in H₂O): 2.2 ´ 10⁴

(195 nm); À2.8 ´ 10⁴(214 nm). IR (KBr): 3290s, 3085w, 2963m, 2940m, 1654s, 1560s, 1458w, 1388w, 1312w,

1

1202m, 1140m, 799w, 722w. H-NMR (500 MHz, CD₃OD): 0.90 ± 0.96 (m, 6 Me); 1.09 ± 1.14 (m, 3 Me); 1.23 ±

2.10 (m, 4 CH, 6 CH₂, Me); 2.18 ± 2.97 (m, 6 CH₂CO, CHCO); 3.54 ± 3.60 (m, CHN); 3.95 ± 4.03 (m, CHN);

4.18 ± 4.26 (m, CHN); 4.37 ± 4.45 (m, CHN); 4.45 ± 4.53 (m, 2 CHN); 4.54 ± 4.63 (m, CHN); 7.51 (br., NH); 7.81

(br., NH); 7.90 (br., NH); 7.93 (br., NH). ¹³C-NMR (125 MHz, CD₃OD): 17.9, 19.2, 19.5, 20.0, 21.1, 21.4, 22.9,

23.1, 23.5, 23.6 (Me); 26.0, 26.1 (CH); 26.2, 26.3 (CH₂); 32.0 (CH); 32.2, 33.2 (CH₂); 34.4 (CH); 36.0, 39.4, 40.6,

42.3, 42.8, 43.1 (CH₂); 43.3, 43.5, 45.4, 45.6 (CH); 45.9, 47.1 (CH₂); 51.1, 52.8, 52.9, 56.0 (CH); 171.2, 171.4, 171.7,

171.8, 173.4, 175.0, 175.7 (C). FAB-MS: 832 (93, [M K] ), 816 (31, [M Na] ),794 (100, [M 1] ).

REFERENCES

[1] a) D. Seebach, M. Overhand, F. N. M. Kühnle, B. Martinoni, L. Oberer, U. Hommel, H. Widmer, Helv.

Chim. Acta 1996, 79, 913; b) D. Seebach, P. E. Ciceri, M. Overhand, B. Jaun, D. Rigo, L. Oberer, U.

Hommel, R. Amstutz, H. Widmer, Helv. Chim. Acta 1996, 79, 2043; c) D. H. Appella, L. A. Christianson,

I. L. Karle, D. R. Powell, S. H. Gellman, J. Am. Chem. Soc. 1996, 118, 13071; d) D. Seebach, S. Abele, K.

Gademann, G. Guichard, T. Hintermann, B. Jaun, J. L. Matthews, J. V. Schreiber, L. Oberer, U. Hommel,

H. Widmer, Helv. Chim. Acta 1998, 81, 932; e) K. Gademann, B. Jaun, D. Seebach, R. Perozzo, L. Scapozza,

G. Folkers, Helv. Chim. Acta 1999, 82, 1; f) T. Sifferlen, M. Rueping, K. Gademann, B. Jaun, D. Seebach,

Helv. Chim. Acta 1999, 82, 2067.

[2] a) S. Krauthäuser, L. A. Christianson, D. R. Powell, S. H. Gellman, J. Am. Chem. Soc. 1997, 119, 11719;

b) Y. J. Chung, L. A. Christianson, H. E. Stanger, D. R. Powell, S. H. Gellman, J. Am. Chem. Soc. 1998, 120,

10555; c) D. Seebach, S. Abele, K. Gademann, B. Jaun, Angew. Chem. 1999, 111, 1700; Angew. Chem., Int.

Ed. 1999, 38, 1595.

[3] T. Hintermann, D. Seebach, Chimia 1997, 51, 244; D. Seebach, S. Abele, J. V. Schreiber, B. Martinoni, A. K.

Nussbaum, H. Schild, H. Schulz, H. Hennecke, R. Woessner, F. Bitsch, Chimia 1998, 52, 734.

[4] a) K. Gademann, M. Ernst, D. Hoyer, D. Seebach, Angew. Chem. 1999, 111, 1302; Angew. Chem., Int. Ed.

1999, 38, 1223; K. Gademann, M. Ernst, D. Seebach, D. Hoyer, Helv. Chim. Acta 2000, 83, 16. b) M.

Werder, H. Hauser, S. Abele, D. Seebach, Helv. Chim. Acta 1999, 82, 1774. c) Y. Hamuro, J. P. Schneider,

W. F. DeGrado, J. Am. Chem. Soc. 1999, 121, 12200; d) E. A. Porter, X. F. Wang, H. S. Lee, B. Weisblum,

S. H. Gellman, Nature 2000, 404, 565.

[5] a) S. Abele, G. Guichard, D. Seebach, Helv. Chim. Acta 1998, 81, 2141; b) B. W. Gung, D. Zou, A. M.

Stalcup, C. E. Cottrell, J. Org. Chem. 1999, 64, 2176; c) D. A. Appella, J. J. Barchi, S. R. Durell, S. H.

Gellman, J. Am. Chem. Soc. 1999, 121, 2309.

[6] A. Jacobi, D. Seebach, Helv. Chim. Acta 1999, 82, 1150.

[7] M. Rueping, hitherto unpublished results, ETH Zürich 1999.

[8] a) H. Estermann, D. Seebach, Tetrahedron Lett. 1987, 28, 3103. b) H. Estermann, D. Seebach, Helv. Chim.

Acta 1988, 71, 1824.

[9] C. K. Johnson, ORTEP II. Report ORNL-5138, Oak Ridge NationalLaboratory, Oak Ridge, Tennessee,

1976.

[10] a) D. Seebach, B. Weidmann, L. Widler, in ꢀModern Synthetic Methods 1983ꢁ, Ed. R. Scheffold, Salle

Sauerländer (Aarau, Switzerland) and J. Wiley and Sons (New York, USA), 1983, Vol. 3, pp. 217 ± 353, and

refs. cit. therein.

[11] a) P. Sieber, Helv. Chim. Acta 1977, 60, 2711. b) H. Gerlach, Helv. Chim. Acta 1977, 60, 3039.

[12] a) D. Seebach, M. Teschner, Tetrahedron Lett. 1973, 51, 5113. b) B. M. Trost, T. N. Salzmann, K. Hiroi, J.

Am. Chem. Soc. 1976, 98, 4887.

[13] D. H. Apella, L. A. Christianson, I. L. Karle, D. R. Powell, S. H. Gellman, J. Am. Chem. Soc. 1999, 121,

6206.

[14] D. Enders, J. Wiedemann, Liebigs Ann./Recueil 1997, 699.

[15] A. Einhorn, A. Meyenberg, Chem. Ber. 1894, 27, 2472.

[16] W. Qiven, A. D. Kline, K. Wütrich, Biochemistry 1987, 26, 6488.

[17] D. Seebach, J. V. Schreiber, S. Abele, X. Daura, W. F. van Gunsteren, Helv. Chim. Acta 2000, 83, 34.

2140

Helvetica Chimica Acta ± Vol. 83 (2000)

[18] M. Carmack, L. A. Neubert, J. Am. Chem. Soc. 1974, 96, 943.

[19] G. Bergson, Ark. Kemi 1958, 12, 233.

[20] H. T. Kalef, E. Havinga, Recl. Trav. Chim. Pays-Bas 1962, 81, 282.

[21] a) M. Carmack, L. A. Neubert, J. Am. Chem. Soc. 1967, 89, 7134; b) G. Claeson, Acta Chem. Scand. 1968,

22, 2429; c) R. M. Dodson, V. C. Nelson, J. Org. Chem. 1968, 33, 3966.

[22] G. Bergson, Ark. Kemi 1962, 18, 409.

[23] J. Linderberg, J. Michl, J. Am. Chem. Soc. 1970, 92, 2619.

[24] a) U. Ludescher, Helv. Chim. Acta 1971, 54, 1637; b) S. Kukolja, P. V. Demarco, N. D. Jones, M. O. Chaney,

J. W. Paschal, J. Am. Chem. Soc. 1972, 94, 7572.

[25] A. Rauk, J. Am. Chem. Soc. 1984, 106, 6517.

[26] L. P. Turchaninova, M. G. Voronkov, A. G. Shipov, N. A. Korchevin, Y. I. Baukov, E. N. Deryagina, Zh.

Obshch. Khim. 1989, 59, 722.

[27] A. L. Davis, E. D. Laue, J. Keeler, D. Moskau, J. Lohman, J. Magn. Reson. 1991, 94, 637.

[28] A. L. Davis, J. Keeler, E. D. Laue, D. Moskau, J. Magn. Reson. 1992, 98, 207.

[29] W. Willker, D. Leibfritz, R. Kerssebaum, W. Bermel, Magn. Reson. Chem. 1993, 31, 287.

[30] C. Griesinger, R. R. Ernst, J. Magn. Reson. 1987, 75, 261.

[31] Sheldrick, G. M. (1993). SHELXS96, University of Göttingen, Germany.

[32] Sheldrick, G. M. (1997). SHELXL97, Program for the Refinement of Crystal Structures, University of

Göttingen, Germany.

[33] a) C. K. Johnson, ORTEPII, Report ORNL-5138. Oak Ridge NationalLaboratory, Tennessee, USA 1976;

b) F. H. Allen, S. Bellard, M. D. Brice, B. A. Cartwright, A. Doubleday, H. Higgs, T. Hummelink, B. G.

Hummelink-Peters, O. Kennard, W. D. S. Motherwell, J. R. Rodgers, D. G. Watson, Acta Crystallogr.,

Sect. B 1976, 35, 2331.

Received May 17, 2000

Article Doi

Synthesis of β-hexa- and β-heptapeptides containing novel β2,3-amino acids with two serine or two cysteine side chains - CD- and NMR-spectroscopic evidence for 314-helical secondary structures in water

DOI: 10.1002/1522-2675(20000906)83:9<2115::AID-HLCA2115>3.0.CO;2-E

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