Optimisation of BACE1 inhibition of tripartite structures by modification of membrane anchors, spacers and pharmacophores - Development of potential agents for the treatment of Alzheimer's disease

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Chemistry

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Volume 10 | Number 41 | 7 November 2012 | Pages 8177–8372

ISSN 1477-0520

PAPER

Hans-Wolfgang Klafki, Hans-Joachim Knölker et al.

Optimisation of BACE1 inhibition of tripartite structures by modiﬁcation of

membrane anchors, spacers and pharmacophores – development of potential

agents for the treatment of Alzheimer’s disease

1477-0520(2012)10:41;1-A

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PAPER

Optimisation of BACE1 inhibition of tripartite structures by modiﬁcation of

membrane anchors, spacers and pharmacophores – development of potential

agents for the treatment of Alzheimer’s disease†

Philipp Linning,^aUte Haussmann,^bIsaak Beyer,^aSebastian Weidlich,^aHeinke Schieb,^bJens Wiltfang,^b

Hans-Wolfgang Klafki*^band Hans-Joachim Knölker*^a

Received 7th June 2012, Accepted 2nd August 2012

DOI: 10.1039/c2ob26103k

Systematic variation of membrane anchor, spacer and pharmacophore building blocks leads to an

optimisation of the inhibitory effect of tripartite structures towards BACE1-induced cleavage of the

amyloid precursor protein (APP).

fragment (sAPPβ).¹¹The residual membrane-tethered C-terminal

fragment C99 is subsequently cleaved by γ-secretase to generate

Introduction

Alzheimer’s disease (AD) is the most common cause of demen-

tia and represents a growing burden for aging societies.¹In

2006, 26.6 million people were estimated to be affected world-

wide by this neurodegenerative disease and projection forecasts

the number of AD patients to rise to over 100 million in 2050.²

According to the amyloid cascade hypothesis,^3,4accumulation

and aggregation of amyloid β-peptides (Aβ-peptides) in the brain

plays a crucial role in the development and progression of AD.

Aβ-peptides are produced by sequential proteolytic cleavages of

the amyloid precursor protein (APP) executed by two mem-

brane-associated enzymes called β- and γ-secretase. The trans-

membrane protein aspartyl protease β-site APP cleaving enzyme

1 (BACE1) (also called Asp2 or memapsin2) was discovered

and cloned by several groups in 1999.^5–9Subsequently, BACE1

was shown to represent the primary β-secretase activity in cul-

tured cells and in vivo.¹⁰BACE1 cleaves APP in the lumenal/

extracellular region to release a soluble, extracellular N-terminal

different Aβ-peptides and the APP intracellular domain (AICD).

BACE1 represents a potential therapeutic target for reducing the

formation of Aβ-peptides. Several BACE1 inhibitors have been

reported.^12–15BACE1 appears to have diverse substrates and

important physiological functions. Therefore, very careful

dosing of BACE1-inhibiting drugs may be required to minimise

possible adverse side effects.¹⁰

Mature BACE1 cycles between the plasma membrane, the

endosomal compartment and the trans-Golgi network. The pro-

teolytic activity of BACE1 on wild-type APP appears to be

mainly conﬁned to the endocytotic pathway^16,17and can be

increased by stimulating BACE1 internalisation.¹⁸Therefore, an

efﬁcient BACE1 inhibitor should be either membrane-permeable

or targeted to the membrane to become available in the endo-

somes. Lipid rafts, liquid-ordered membrane domains enriched

in cholesterol and sphingolipids,^19,20appear to be important for

BACE1 cleavage of APP. BACE1 has been detected in lipid raft

fractions²¹and reduction of cholesterol was shown to decrease

cellular Aβ-production.^22,23

In order to achieve high local concentrations of BACE1

inhibitors in designated subcellular regions,¹⁶we have reported

the “tripartite structure” concept in 2005.²⁴Tripartite structures

are assembled by covalent coupling of BACE1 inhibitors with

membrane anchors via suitable spacers. The spacer is designed

to keep the inhibitor at an appropriate distance from the plasma

membrane. The membrane anchor should bind the tripartite

structure preferentially to the lipid raft domains of the membrane.

Thus, an enrichment of the tripartite structure in the lipid rafts is

achieved with “raftophilic” membrane anchors (Fig. 1).

^aDepartment Chemie, Technische Universität Dresden, Bergstrasse 66,

01069 Dresden, Germany. E-mail: hans-joachim.knoelker@tu-dresden.de

^bLVR Klinikum Essen, Klinik für Psychiatrie und Psychotherapie,

Universität Duisburg-Essen, Virchowstrasse 171, 45147 Essen,

Germany. E-mail: hans.klafki@uni-due.de

†Electronic supplementary information (ESI) available: Analytical

HPLC chromatograms, low resolution and high resolution ESI mass

1

spectra (and H and ¹³C NMR spectra) of 6a–6j, 7a–7q, 9–11, 13, 14,

16, 18–20; ¹H NMR spectra for 33 and 41; ¹H and ¹³C NMR spectra for

24, 28, 32, 47 and 48; log EC₅₀values and SD for all tested compounds;

for tripartite structures with different membrane anchors: comparison of

EC₅₀values calculated by averaging log EC₅₀values of three or more

independent experiments and EC₅₀values calculated from non-linear

regression of pooled data and pairwise comparison of log EC₅₀values

by ANOVA and Tukey’s post-test; detailed statistical comparison of the

tripartite structures 19 and 20 with the pharmacophores 17, 50 and 51.

See DOI: 10.1039/c2ob26103k

Tripartite structures are rapidly internalised by endocytosis

and inhibit BACE1 much more efﬁciently than the correspond-

ing free inhibitors in cell lines and primary neuronal cell

culture.^25,26Moreover, they were reported to be active in vivo

8216 | Org. Biomol. Chem., 2012, 10, 8216–8235

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Scheme 1 Synthesis of membrane anchor building blocks 3a–j.

Reagents and conditions: (a) 1.1 equiv. Fmoc-Asp-OtBu (1), 1.0 equiv.

Dhc-OH (or Chol-OH, C_nH_2n+1OH (n = 14, 16, 18, 20, 22, 24, 26) or

oleyl-OH), 1.1 equiv. MSNT and 1.1 equiv. MeIm (or 1.5 equiv.

EDC·HCl cat. DMAP), CH₂Cl₂, rt, 4–52 h; (b) CF₃COOH, CH₂Cl₂

(1 : 0.8–1.5), rt, 90 min, 3a: 71%, 3b: 61%, 3c: 67%, 3d: 64%, 3e: 76%,

3f: 68%, 3g: 92%, 3h: 70%, 3i: 80%, 3j: 65% (yields over two steps).

Fig. 1 Tripartite structure concept. Anchoring of the pharmacophore

(BACE1 inhibitor) via an appropriate spacer to a raftophilic membrane

anchor leads to an increase of the inhibitor concentration in the proxi-

mity of the active site of BACE1 as both are located in the lipid rafts.

when injected stereotactically into the hippocampus of transgenic

APPsw/PSΔ9 mice.²⁵

Herein, we describe the synthesis of novel tripartite structures

and extensive studies regarding their structure–activity relations

(SAR) in cell-based assays. In continuation of our previous

work,^24–26we report in detail the effect of different lipophilic

membrane anchors and spacers on the activity of tripartite struc-

tures carrying the prototype peptidic pharmacophore GL189.

Furthermore, we show that the potency of different non-peptidic

transition state BACE1 inhibitors is substantially improved by

incorporation into our tripartite structures.

Fig. 2 Building blocks for the spacer moiety of the tripartite structures.

Results and discussion

aminomethylphenylacetic acids 5a and 5b were tested as alterna-

tive building blocks. Using the commercially available N-Fmoc-

ω-amino(oligoethylene glycol)carboxylic acids 4a–g or the

N-Fmoc-aminomethylphenylacetic acids 5a and 5b as building-

blocks, spacers of various lengths were prepared. The N-Fmoc-

protected carboxylic acids were directly suitable for solid-phase

peptide synthesis (Fig. 2).

The tripartite structures were prepared by automated solid-

phase peptide synthesis and puriﬁed by preparative HPLC (see

the Experimental section). The structures were supported by

mass spectrometry. Previous advanced NMR experiments

conﬁrmed that the peptide synthesis proceeded without epimeri-

sation and single diastereoisomers were obtained.²⁶According to

HPLC analysis, the purities of tripartite structures generally were

higher than 99.9% (Table 1).

Tripartite structures were assembled from preformed building

blocks by solid-phase peptide synthesis. The building blocks,

used as pharmacophores, spacers and lipid anchors, were either

obtained commercially or synthesised as described below. For a

detailed SAR study by variation of lipid anchor and spacer

modules, we used the same peptidic substrate-based inhibitor as

before (GL189 = H-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-NH₂).²⁷

Modiﬁcations of further literature-known BACE1 inhibitors,

obtained by de novo synthesis, provided additional building

blocks for solid-phase peptide synthesis.

The membrane anchor building blocks were obtained by esteri-

ﬁcation of lipidic alcohols, e.g. dihydrocholesterol (Dhc-OH)

with the protected amino acid Fmoc-Asp-OtBu (1) using 1-(2-

mesitylensulfonyl)-3-nitro-1H-1,2,4-triazole (MSNT) or N-(3-

As described previously, the biological activity of the tripartite

structures was determined by measuring the concentration of

Aβ40 peptides secreted by cultured SH-SY5Y cells overexpres-

sing human wild-type APP695.²⁶

dimethylaminopropyl)-N′-ethylcarbodiimide

hydrochloride

(EDC·HCl) (Scheme 1). Cleavage of the tert-butyl esters 2a–j

with triﬂuoroacetic acid afforded the Fmoc-protected building

blocks 3a–j, suitable for solid-phase peptide synthesis.

Ideally, the inhibitor should be positioned by the spacer close

to the active site of BACE1 by keeping a similar distance from

the plasma membrane. Moreover, the spacer should not affect

the interaction between BACE1 and the inhibitor. We have

chosen oligoethylene glycol units as spacer modules due to their

low toxicity, high bio-compatibility, high stability and good solu-

bility under physiological conditions. To investigate the potential

inﬂuence of the chemical composition of spacer modules, the

Variation of the membrane anchor

The afﬁnity of membrane anchors to lipid rafts depends on their

structural features. Therefore, we have studied the effect of

different membrane anchors on the efﬁciency of tripartite struc-

tures as BACE1 inhibitors in a cellular assay. Assuming that

amyloidogenic APP cleavage by BACE1 is dependent on lipid

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Table 1 Analytical data for the tripartite structures 6a–j, 7a–q, 9–11,

13, 14, 16 and 18–20

No. Retention time^a[min] Purity^b[%] m/z^c(calcd) m/z^d(found)

6a

6b

6c

6d

6e

6f

6g

6h

6i

27.5

26.5

20.4

22.3^e

24.7

27.2

29.4

31.8

34.3

23.1^e

29.7

29.2

30.0

27.8

27.9

29.8

27.1

27.6

28.2

19.5^f

20.1^f

29.1^e

>99.9

99.1

>99.9

99.9

>99.9

99.9

2595.5516

2593.5360

2421.4108

2449.4421

2477.4734

2505.5047

2533.5360

2561.5673

2589.5986

2475.4577

1783.0886

1854.1257

1871.1410

1898.1519

2057.2415

2047.2459

2101.2677

2118.2830

2145.2939

2295.3310

2189.3201

2223.3507

2260.3572

2436.4621

2575.5605

2683.6041

2210.3054

2254.2952

2180.2796

1970.0828

1984.0985

2192.3966

2105.3042

2210.3620

2195.3511

2595.5529

2593.5356

2421.4113

2449.4423

2477.4737

2505.5050

2533.5346

2561.5669

2589.6005

2475.4522

1783.0863

1854.1267

1871.1423

1898.1530

2057.2411

2047.2499

2101.2650

2120.0^g

6j

7a

7b

7c

7d

7e

7f

7g

7h

7i

99.4

99.9

>99.9

97.8

>99.9

99.4

>99.9

99.1

Fig. 3 Selected dose–response curves for quantifying the inhibition of

Aβ40 secretion by tripartite structures with different membrane anchors.

Aβ40 concentrations in cell culture supernatants are expressed as percent

of the control condition C (1% DMSO). Compounds 6a and 6b were

tested at concentrations ranging from 10 p^Mto 100 nM in SH-SY5Y

APPwt cells. 6c and 6i were tested at concentrations from 1 n^Mto

10 μ^M; 6d was tested with alternating concentration ranges (once:

100 p^Mto 1 μM; twice: 1 nM to 10 μM); 6f was tested at concentrations

from 100 p^Mto 1 μM. The dose–response curves were generated by

pooling normalised data (means and SD) from three independent

experiments (ESI, Table S2†). It should be noted that the concentration

ranges were varied between experiments for some compounds. In cases

where error bars are shown, they represent data from at least two

independent experiments.

2145.2938

7j

7k

7l

2297.4^g

2295.3295

2189.3208

2223.3509

2260.3574

2436.4628

2575.5616

2683.6069

2211.8^g

2254.2970

2180.2792

1970.0832

1984.0994

2192.3962

2105.3043

2210.3630

2195.3513

7m 29.0

7n

7o

7p

7q

9

10

11

13

14

16

18

19

20

27.5

27.7

28.8

27.3

34.2

32.9

30.4

32.9

33.1

30.8

27.3

27.0

27.6

99.8

99.6

98.8

>99.9

99.3

>99.9

99.9

>99.9

Swedish-mutant or wild-type APP showed differences with

respect to the BACE1 inhibiting activity of tripartite structures

and the secretion of Aβ-peptides.²⁶Therefore, we conducted an

extensive comparative study of the activity of tripartite structures

with a range of different membrane anchors (long-chain lipid

alcohols ranging from C₁₄–C₂₆, dihydrocholesterol and choles-

terol) in SH-SY5Y cells overexpressing wild-type APP. To

compare the tripartite inhibitors on the basis of reliable EC₅₀

values, each compound was tested in serial dilutions in at least

three independent experiments. In addition, for some compounds

the range of tested concentrations was varied between different

experiments to improve the accuracy of the determination of top

and bottom plateaus of the dose–response curves. Consequently,

not each single compound dilution depicted below was tested in

all three experiments. The dose–response curves for tripartite

compounds 6a–d, 6f and 6i are shown in Fig. 3.

^aHPLC retention time: column: Vydac 208TP104 (reverse-phase C₈, 4.6

× 250 mm). Flow rate: 1.0 mL min⁻¹. Eluent A: H₂O + 0.1%

CF₃COOH, eluent B: MeCN + 0.1% CF₃COOH. Gradient from 20% to

90% B in 35 min. ^bAnalysis by an evaporative light scattering detector.

^cMonoisotopic mass. ^dReconstructed monoisotopic mass. ^eEluent A:

H₂O/MeCN (85 : 15) + 0.1% CF₃COOH, eluent B: MeCN + 0.1%

f

CF₃COOH. Gradient from 10% to 100% B in 45 min. Eluent A: H₂O/

MeCN (85 : 15)

+ 0.1% CF₃COOH, eluent B: MeCN + 0.1%

CF₃COOH. Gradient from 30% to 100% B in 35 min. ^gMost abundant

mass observed for the adduct [M + H]⁺.†

rafts, tripartite structures with very efﬁcient raftophilic membrane

anchors should exhibit the highest potency. The tripartite struc-

tures 6a–j (Table 1 and 2) were synthesised using the membrane

anchor building blocks 3a–j (Scheme 1), the peptidic inhibitor

GL 189 and the 4Gl-3Gl-4Gl-3Gl-4Gl spacer module.

The spacer length was kept constant (approx. 89 Å, assuming

an extended conformation of the spacer) to have results compar-

able with existing data.²⁶The effect of 6a, 6c, 6d and 6j on the

generation of sAPPβ by HeLa cells overexpressing the Swedish-

mutant form of human APP was studied before at two concen-

trations (20 and 200 n^M).²⁵The tripartite structure 6a with the

dihydrocholesteryl anchor was reported to be most potent. Com-

pound 6j with a cis double bond in the oleyl anchor was found

to be essentially inactive. Overall, the efﬁciency of different tri-

partite structures in BACE1 inhibition appeared to resemble their

degree of raft partitioning. In our previous study, cells expressing

The tripartite structures 6a and 6b with cholesterol and dihy-

drocholesterol as raftophilic membrane anchors reduced very

efﬁciently Aβ40 release into the cell culture medium. The EC₅₀

values of 0.39 (log EC₅₀= −9.406 0.182) and 0.74 nM (log

EC₅₀= −9.130 0.035) calculated for 6a and 6b were substan-

tially lower than those of the tripartite structures 6c–i with long-

chain lipid alcohols as membrane anchors (Table 2). To evaluate

the statistical signiﬁcance of the observed differences, we com-

pared the log EC₅₀values by one-way ANOVA and Tukey’s mul-

tiple comparison post-test. The difference in log EC₅₀between

6a (dihydrocholesteryl anchor, Dhc) and 6b (cholesteryl anchor,

8218 | Org. Biomol. Chem., 2012, 10, 8216–8235

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Table 2 Tripartite structures 6a–j with different membrane anchors

No.

Sequence^a

EC₅₀^b[nM]

6a

6b

6c

6d

6e

6f

0.39

0.74

177.01

24.60

9.16

6.82

6g

6h

6i

7.24

7.66

23.39

n.d.^c

6j

Fig. 4 Selected dose–response curves. Inhibition of Aβ40 secretion by

tripartite structures with different lipid alcohol anchors. Aβ40 concen-

trations in cell culture supernatants are expressed as percent of the

control condition C (1% DMSO). Compound 6e was tested with alterna-

ting concentration ranges (once: 1 n^M–10 μM; twice: 100 pM–1 μM); com-

pounds 6f and 6h were tested at concentrations ranging from 100 p^Mto

1 μ^Min SH-SY5Y APPwt cells. Compound 6g was tested twice at nine

different concentrations from 100 p^M–1 μM, while in the third exper-

iment, a reduced number of only six concentrations was tested, ranging

from 3 n^Mto 1 μM. Dose–response curves were generated by pooling

normalised data (means and SD) from three independent experiments.†

^aInhibitor: H-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe; spacer: 4Gl-3Gl-4Gl-

3Gl-4Gl. ^bHalf maximal effect (EC₅₀) of inhibition of Aβ40 secretion

from treated cells relative to control (1% DMSO). Log EC₅₀was

determined by non-linear regression from dose–response curves. The

value given is the delogarithmised mean value of three independent

experiments (ESI†). ^cn.d. = not determined, 6j was inactive at all

concentrations tested (up to 100 n^M).

Chol) did not reach statistical signiﬁcance. However, both were

signiﬁcantly lower than each single log EC₅₀value obtained for

6c–6i (for pairwise comparison of different anchors, see ESI,

Table S1†). This result is explained by the different partitioning

of sterol and lipid alcohol anchors between raft and non-raft

phases. As dihydrocholesteryl anchors bind more efﬁciently to

the raft phase,²⁵the tripartite structures 6a and 6b should be

available in higher concentrations at the subcellular site of amy-

loidogenic APP cleavage. Within the series of tripartite structures

with lipid alcohol anchors, a chain length of 18 to 24 carbon

atoms provided the best result. Compounds 6e–h were equally

potent (Fig. 4), whereas the log EC₅₀of 6c (log EC₅₀= −6.752

0.266) with the shortest lipid chain (C₁₄) was signiﬁcantly

higher than all other log EC₅₀values in this series (p < 0.001;

ESI, Table S1†). Compared to compounds 6e–h, the potency

appeared to decrease for 6d (log EC₅₀= −7.609 0.342) and 6i

(log EC₅₀= −7.631 0.124) with chain lengths of 16 and 26

atoms, albeit statistical signiﬁcance was not reached. It is

assumed that the observed inﬂuence of the chain length within

lipid alcohol anchors may reﬂect subtle differences in the distri-

bution between raft and non-raft domains.

shown to efﬁciently reduce cellular Aβ production (compound

6a with an 89 Å spacer displayed the lowest EC₅₀value).²⁶To

extend our previous work and to investigate the inﬂuence of

both, spacer length and chemical composition, we tested a series

of tripartite structures with theoretical spacer lengths ranging

from 26 to 96 Å. The subtle variations in length in this range

were implemented by different combinations of the spacer build-

ing blocks 4a–c, thus providing eleven tripartite structures: 6a,

7b, 7d, 7e, 7g–i, 7l, 7n, 7o and 7q (Table 1 and 3).

Spacers with different chemical entities were tested. Pre-

viously, different spacer lengths ranging from 35 to 123 Å were

assembled by amide coupling of the ω-aminooligoethylene

glycol carboxylic acids 4a and 4b. In compounds 7a, 7c, 7f, 7m

and 7p one-block oligoethylene glycol units have been used as

spacers. They derive from the ω-aminooligoethylene glycol car-

boxylic acids 4c–g containing 6, 8, 12, 16 and 24 oligoethylene

glycol units and thus have no amide bonds within the spacer

module. The phenylacetic acids 5a and 5b were used to construct

a further type of spacer and led to the tripartite structures 7j and

7k with more lipophilic aromatic moieties in the spacer module

(Fig. 2).

Variations of the spacer module

According to the original tripartite structure concept,²⁴the

length of the spacer linking the pharmacophore to the membrane

anchor was expected to play a crucial role for the inhibitory

activity. The optimum spacer should position the pharmacophore

at a distance from the plasma membrane approximately matching

that of the BACE1 active site (or that of the β-secretase cleavage

site within APP) (Fig. 1). Theoretical spacer lengths of our tri-

partite inhibitors have been estimated assuming an all-anti-con-

formation for the chain. Therefore, these lengths correspond to a

maximal theoretical length irrespective of the actual confor-

mation of the spacer. In a previous study, tripartite structures

with theoretical spacer lengths ranging from 35 to 89 Å were

In order to get data which are comparable to the previous

ones, dihydrocholesterol was used as a lipid anchor and the pep-

tidic transition state inhibitor GL189 as a pharmacophore. The

tripartite structures 6a and 7a–q were shown to be highly active

(Table 3). Irrespective of the chemical composition of the spacer

and the theoretical length (ranging from 26–96 Å), 6a and 7a–q

exhibited EC₅₀values below 7 nM. This ﬁnding indicates that

within these limits the activities of our tripartite structures are

relatively independent of the structure of the spacer module.

As shown in Fig. 5, the log EC₅₀values of the tripartite struc-

tures 6a and 7a–q did not show a single minimum. The com-

pounds could be roughly divided into two groups with EC₅₀

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Table 3 Tripartite structures 6a and 7a–q with different spacers

Spacer

length^b

[Å]

c

EC₅₀

[nM]

No. Sequence^a

7a

7b

7c

7d

7e

7f

26

31

33

35

47

48

51

53

57

58

61

63

77

89

91

96

0.56

0.53

0.43

2.64^d

1.11^d

0.37

0.51

0.52

0.44

0.45

0.59

0.81

6.18

1.99^d

0.67

0.39^d

0.79

1.55

7g

7h

7i

Fig. 5 Dependency of the log EC₅₀from the theoretical spacer length

for tripartite structures with oligoethylene glycol spacers. Compounds

7b, 7d, 7e, 7g–7l, 7n, 7o and 7q with assembled spacers are depicted as

blue dots. Compounds 7a, 7c, 7f, 7m and 7p with one-block spacers

without internal amide bonds are depicted as red squares. To assess the

statistical signiﬁcance, the mean log EC₅₀values were compared to that

of compound 6a (green dot) by one way-ANOVA combined with Dun-

nett’s multiple comparison post-test. The mean log EC₅₀values of com-

pounds 7d, 7e, 7m, 7n and 7q were signiﬁcantly different from that of

6a (**p < 0.01; ***p < 0.001) and higher than −9 (EC₅₀= 1 nM).

7j

7k

7l

7m

7n

7o

6a

7p

7q

(log EC₅₀= −8.954 0.318), 7m (log EC₅₀= −8.209 0.216),

7n (log EC₅₀= −8.702 0.252) and 7q (logEC₅₀= −8.810

0.055) were signiﬁcantly different from that of 6a (log EC₅₀

−9.406 0.182).

=

^aInhibitor: H-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe; raftophile: Asp

(ODhc)-NH₂. ^bMaximal theoretical spacer length, assuming an all-anti

conformation. ^cHalf maximal effect (EC₅₀) of inhibition of Aβ40

secretion from treated cells relative to control (1% DMSO). The value

given is the delogarithmised mean log EC₅₀of three independent

experiments, except for 6a, which was tested in eight independent

experiments. All compounds were tested at concentrations from 10 p^Mto

100 n^M, except for 7m, which was tested at alternating ranges (once:

10 p^M–100 nM; twice: 100 pM–1 μM). Log EC₅₀was determined by non-

linear regression from dose–response curves (ESI, Table S2†). ^dEC₅₀

values of compounds 6a, 7d, 7e and 7n were also reported in ref. 26.

Subtle differences are due to adjustments in the statistical analysis of the

data allowing direct comparison (ESI, Table S2†).

Apparent minima of the EC₅₀values were observed for tripar-

tite structures with theoretical spacer lengths of 33 Å (7c: EC₅₀

= 0.43 nM; log EC₅₀= −9.368 0.228), 48 Å (7f: EC₅₀= 0.37

n^M; log EC₅₀= −9.436 0.038) and 89 Å (6a: EC₅₀= 0.39 nM;

log EC₅₀= −9.406 0.182). However, it should be noted that

the subtle differences of activity of tripartite structures with EC₅₀

values <1 nM did not reach statistical signiﬁcance. In summary,

our results indicate that the relative potency of the tripartite struc-

tures with theoretical spacer lengths ranging from 26 to 96 Å did

neither solely depend on the number of amide groups nor on the

chemical composition of the spacers. Oligoethylene glycols can

form helical structures in water,²⁸and thus it does not seem

likely that the spacer molecules adopt a fully extended confor-

mation in a physiological environment. Interactions with the

aqueous medium and with cell membrane-related components

may inﬂuence the conformation of the spacer and thus affect the

potency of the tripartite structure.

values below and above 1 nM, respectively. Interestingly, the

difference in potency does not seem to depend on the chemical

composition of the spacer. Among the structures with one-block

oligoethylene glycol spacers, the four compounds 7a, 7c, 7f and

7p exhibit EC₅₀values < 1 nM (7a: EC₅₀= 0.56 nM; log EC₅₀

−9.248 0.097; 7c: EC₅₀= 0.43 nM; log EC₅₀= −9.368

=

0.228; 7f: EC₅₀= 0.37 nM; log EC₅₀= −9.436 0.038; 7p:

EC₅₀= 0.79 nM; log EC₅₀= −9.104 0.113), whereas the value

Variation of the BACE1 inhibitor

of compound 7m was much higher (EC₅₀= 6.18 nM, log EC₅₀

=

Membrane anchoring considerably increases the efﬁcacy of the

peptidic BACE1 inhibitor GL189 in cellular assays.^24–26In the

present study, the tripartite structure concept has been tested on

four literature-known BACE1 inhibitors: 8, 12, 15 and 17

(Fig. 6).^29–32The inhibitors were adapted for solid-phase peptide

synthesis to enable the attachment of a spacer and a raftophile.

Thus, the structures had to be modiﬁed by adding a carboxyl

group which served as a coupling partner for the N-terminus of

the spacer. The modiﬁed pharmacophores were synthesised fol-

lowing literature procedures.^29–32The additional carboxyl groups

either derive from modiﬁed protected starting materials or from

attachment of carboxyl group-bearing moieties at a later stage of

−8.209 0.216). Spacer modules containing phenylacetic acid

as in 7j and 7k did not affect the activity of the tripartite struc-

tures as compared to spacers consisting exclusively of oligoethy-

lene glycol units with theoretical spacer lengths from 48 Å to

58 Å (7j: EC₅₀= 0.45 nM; log EC₅₀= −9.343 0.080; 7k: EC₅₀

= 0.59 nM; log EC₅₀= −9.229 0.282) (Table 3). For a statisti-

cal evaluation of the results, 6a was chosen as the reference. The

mean log EC₅₀values of each spacer variation were compared to

the mean log EC₅₀of 6a by one-way ANOVA followed by Dun-

nett’s multiple comparison post-test. The mean log EC₅₀values

of the tripartite structures 7d (log EC₅₀= −8.578 0.327), 7e

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Fig. 6 BACE1 inhibitors 8, 12, 15, 17 and tripartite analogues 9–11, 13, 14, 16, 18–20.

-4Gl-3Gl-4Gl-3Gl-4Gl-Asp(ODhc)-NH₂.

EC₅₀: inhibitor concentration at which inhibition of Aβ40 secretion from treated cells was half-maximal compared to the control condition (1%

DMSO). The value given is the delogarithmised mean log EC₅₀of three independent experiments. Log EC₅₀was determined by non-linear regression

from dose–response curves (ESI†).

the synthesis. Thus, the synthetic routes were kept similar to the

original procedures and the building blocks for the solid-phase

synthesis were obtained in a convergent manner.

different coupling strategy might lead to active tripartite deriva-

tives of 8, we decided to connect the inhibitor and the spacer

either via modiﬁcation of the p-toluenesulfonamide or by repla-

cement of the Fmoc-protecting group by a phenylacetic acid

derivative. Both analogues could still exhibit inhibitory activity

based on the structure–activity study.²⁹The building blocks 24

and 28 for the solid-phase peptide synthesis were prepared from

H-Lys(Cbz)-OtBu (21) (Scheme 2). For the synthesis of com-

pound 24 with the modiﬁed sulfonamide, amine 21 was reduc-

tively alkylated with isobutyraldehyde. The secondary amine

was converted into the sulfonamide 22 using benzyl 2-(4-(chloro-

sulfonyl)phenyl)acetate, which was prepared from 4-iodopheny-

lacetic acid via benzyl protection, copper-catalysed coupling

with thiobenzoic acid³³and subsequent oxidative chlorination

with trichloroisocyanuric acid.³⁴Deprotection of 22 by cleavage

of the benzyl ester and the carbamate followed by Fmoc-protec-

tion of the amine 23 afforded building block 24.

The ﬁrst small-molecule inhibitor examined was the lysine

derivative

8 (Elan Pharmaceuticals/Pharmacia & Upjohn;

Fig. 6).^13,29A K_ivalue of 5 nM was reported for that compound.

Tripartite structure 9 was obtained by coupling of the free car-

boxylic acid 8 to the spacer–membrane anchor module (-4Gl-

3Gl-4Gl-3Gl-4Gl-Asp(ODhc)-NH₂). The tripartite structure 9

was reported to be an active inhibitor of BACE1 in cell-based

assays.²⁶This ﬁnding could not be reproduced and our present

study demonstrated that the previous result was caused by an

impurity.

In a previous structure–activity study, the isobutyl group at the

sulfonamide and the free carboxylic acid were found to be

crucial for the inhibitory effect of 8.²⁹Thus, the carboxylic acid

group of compound 8 should not be functionalised. As a

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Scheme 3 Synthesis of the building blocks 32 and 33. Reagents and

conditions: 1.0 equiv. 30 or 31, 1.0 equiv. 29, 0.85 equiv. AcOH, EtOH,

78 °C, 2.5 h, 66% for 32, 51% for 33.

available 4-hydrazinobenzoic acid (30) and 4-hydrazinophenyl-

acetic acid (31), prepared by diazotation and reduction of

4-aminophenylacetic acid.³⁷

Acid-catalysed condensation of ketone 29 with the hydrazines

30 and 31 provided the building blocks 32 and 33 with no need

for protecting group operations at the carboxylic acids.³⁸The tri-

partite structures 13 and 14, as well as the inhibitors 32 and 33

were unable to reduce the Aβ40-secretion from SH-SY5Y

APPwt cells at concentrations of up to 100 n^M(Fig. 6).

The third non-peptidic small-molecule inhibitor of BACE1

investigated in our present study was the 2-amino-3,4-dihydro-

quinazoline 15 (Johnson & Johnson; Fig. 6).³¹An X-ray analysis

of a structurally related 2-amino-3,4-dihydroquinazoline bound

to the active site of BACE1 showed that the primary amine inter-

acts with the catalytic aspartates of BACE1.³¹A K_ivalue of 30

n^Mwas found for rac-15, which was also active in a cell-based

assay. Attachment of this inhibitor to the tripartite structure was

achieved by substitution of the phenoxy group at the 6-position

of the 3,4-dihydroquinazoline 15 by a 4-hydroxyphenylacetic

acid. The primary amine and the cyclohexyl moieties are impor-

tant for the activity of 15 because they interact with the catalyti-

cally active aspartates or the S₁and S₁′ pockets of BACE1,

respectively. Therefore, these structural units were left

untouched. Furthermore, the modiﬁed substituent at the 2-

amino-3,4-dihydroquinazoline could easily be introduced at the

beginning of the synthesis, keeping the remaining steps similar

to the known procedure (Scheme 4).³¹4-Hydroxyphenylacetic

acid (34) was converted to the tert-butyl ester 35 using an excess

of tert-butyl acetoacetate.³⁹Nucleophilic aromatic substitution

by reaction of 5-ﬂuoro-2-nitrobenzaldehyde with the phenol 35

provided the diaryl ether 36. Subsequent reductive amination

with amine 37 afforded compound 38.³¹Reduction of 38 to the

aniline 39 followed by cyclisation using cyanogen bromide pro-

vided the 2-amino-3,4-dihydroquinazoline 40. Cleavage of the

tert-butyl ester led to the phenylacetic acid 41.⁴⁰

Scheme 2 Synthesis of the building blocks 24 and 28. Reagents and

conditions: (a) 1.14 equiv. isobutyraldehyde, 0.66 equiv. EtN(i-Pr)₂,

1.14 equiv. NaBH₃CN, 2.4 equiv. AcOH, MeOH, rt, 2 h, 92%; (b) 1.0

equiv. benzyl 2-(4-(chlorosulfonyl)phenyl)acetate, 1.0 equiv. EtN(i-Pr)₂,

CH₂Cl₂, rt, 3 d, 63%; (c) cat. 10% Pd/C, H₂(1 atm), EtOAc, rt, 23 h,

95%; (d) 1.5 equiv. N-(FmocO)succinimide, MeCN, THF, 1 ^MK₂CO₃in

H₂O (1 : 1 : 1), rt, 17 h, 51%; (e) 1.0 equiv. TosCl, 1.5 equiv. EtN(i-Pr)₂,

CH₂Cl₂, rt, 5 d, 88%; (f) cat. 10% Pd/C, H₂(1 atm), EtOAc, rt, 24 h,

99%; (g) 1.0 equiv. 26, 1.5 equiv. 27, 1.5 equiv. EDC·HCl, cat. DMAP,

CH₂Cl₂, rt, 5 d, 56% (h) cat. 10% Pd/C, H₂(1 atm), EtOAc, rt, 2 h,

100%.

Reductive amination of compound 21 and subsequent reaction

with 4-toluenesulfonyl chloride afforded the sulfonamide 25.²⁹

Deprotection of the primary amine led to compound 26, which

on amidation with the acid 27 and cleavage of the benzyl ester

provided building block 28. Acid 27 is accessible by chemo-

selective protection of 4-hydroxyphenylacetic acid (34) as

benzyl ester,³⁵reaction with tert-butyl bromoacetate and

cleavage of the tert-butyl ester.

The free inhibitor 8 and the derived tripartite structures 9, 10

and 11 were all unable to inhibit the Aβ40-secretion from

SH-SY5Y APPwt cells at concentrations of up to 100 n^M(8) and

1 μ^M(9, 10 and 11), respectively.

As the second small-molecule inhibitor we have chosen the

hydrazone 12 (The Genetics Company Inc., Switzerland;

Fig. 6).³⁰An IC₅₀value of 14.4 μM for an in vitro assay and

activity in a cell-based assay were reported.^30,36Since no infor-

mation on structure–activity relations was available, this inhibitor

was attached via the mono-substituted aromatic ring using a

benzoic or phenylacetic acid moiety (Scheme 3). The synthesis

of the building blocks 32 and 33 started from commercially

The tripartite structure 16 turned out to be a potent inhibitor

of cellular Aβ release from SH-SY5Y APPwt cells. The EC₅₀of

14.8 n^M(log EC₅₀= −7.831 0.057) for 16 as compared to

5.4 μ^M(log EC₅₀= −5.266 0.060) for the pharmacophore 41

indicated a substantial improvement of potency (Fig. 7). A sig-

niﬁcant statistical difference for the mean log EC₅₀values of 16

and 41 was revealed by Student’s t-test (p < 0.0001). This result

showed that membrane targeting of a non-peptidic compound,

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Scheme 4 Synthesis of building block 41. Reagents and conditions:

(a) 6.4 equiv. tert-butyl acetoacetate, 0.08 equiv. conc. H₂SO₄, closed

vessel, rt, 45 h, 41%; (b) 1.0 equiv. 5-ﬂuoro-2-nitrobenzaldehyde, 1.2

equiv. K₂CO₃, DMF, 120 °C, 45 min, 63%; (c) 1.0 equiv. 36, 1.0 equiv.

37, 1.1 equiv. NaBH₃CN, 1.0 equiv. AcOH, CH₂Cl₂, rt, 17 h, 83%; (d)

cat. 10% Pd/C, H₂(1 atm), MeOH, rt, 2 h, 94%; (e) 1.5 equiv. BrCN,

EtOH, 78 °C, 17 h, 84%; (f) 8.2 wt-equiv. silica gel, toluene, 111 °C,

3.5 h, 99%.

Fig. 7 (A) Dose–response curves: inhibition of Aβ40 secretion by tri-

partite structure 16 (red dots) and pharmacophore 41 (black squares).

Aβ40 concentrations in cell culture supernatants are expressed as percent

of the control condition C (1% DMSO). The curves were generated by

non-linear regression on pooled normalised data from three independent

experiments. Compound 16 was tested in alternating concentration

ranges in SH-SY5Y APPwt cells (once: 30 p^M–100 nM, 1 μM; twice:

300 p^M–3 μM). Compound 41 was tested in two experiments at nine different

concentrations ranging from 10 n^Mto 100 μM and for conﬁrmation, in a

third experiment at a reduced number of concentrations (10 n^M, 100 nM,

1 μ^M, 10 μM, 100 μM). (B) Log EC₅₀values of tripartite structure 16 and

pharmacophore 41 from three independent experiments were compared

by an unpaired, two-tailed t-test. Mean log EC₅₀values were signiﬁ-

cantly different (***p < 0.0001). The lines and error bars represent

means and standard deviations from three independent experiments.

which is only weakly active in a cellular assay, leads to a signiﬁ-

cant increase of the inhibitory activity.

As the fourth small-molecule BACE1 inhibitor we tested the

spiropiperidine iminohydantoin 17 which was identiﬁed by

high-throughput screening (Merck; Fig. 6).³²An IC₅₀value of

2.8 μ^Mon the isolated enzyme and an activity in a cellular assay

at 8.2 μ^Mhave been reported.³²The cyclohexyl and ﬂuorophenyl

rings of 17 were found to be essential for high potency in vitro

and good activity in the cell-based assay, respectively. Therefore,

attachment to the spacer was achieved by modiﬁcation of the

substituent at the piperidine nitrogen. The spiropiperidine 42 is

readily available via an Ugi reaction (Scheme 5).³²In a ﬁrst

attempt the piperidine nitrogen of 42 was directly acylated with

Fmoc-Glu-OtBu (43). Subsequent acidic cleavage of the tert-

butyl ester afforded building block 44. For the two pharmaco-

phores 50 and 51, attachment to the spacer was projected by sub-

stitution of the 4-acetamido group of 17 with a glutamic or

glutaric acid linkage, respectively. Thus, the structure of the

pharmacophores was kept similar to the one reported in the lit-

erature. Protection of 4-nitrobenzyl alcohol by reaction with tert-

butylchlorodimethylsilane, reduction to the aniline,⁴¹amidation

with either Fmoc-Glu-OtBu (43) or mono-tert-butyl glutarate,⁴²

removal of the silyl protecting group and oxidation with manga-

nese dioxide^43,44afforded the benzaldehydes 45 and 46.

Reductive amination of the spiropiperidine 42 with the benzal-

dehydes 45 and 46 and subsequent cleavage of the tert-butyl

ester led to compound 49 and the pharmacophore 50. Cleavage

of the Fmoc group in 49 provided the glutamic acid-derived

pharmacophore 51.

The tripartite structures 19 and 20 exhibited potent inhibition

of Aβ40-secretion in a cellular assay with EC₅₀values of 50.1 nM

(log EC₅₀= −7.300 0.163) and 9.7 nM (log EC₅₀= −8.014

0.048) (Fig. 6), whereas the reference substances 17, 50 and

51 showed considerably higher EC₅₀values of 0.863 μM (log

EC₅₀= −6.064 0.178), 17.5 μM (log EC₅₀= −4.758 0.212)

and 4.3 μ^M(log EC₅₀= −5.368

0.150), respectively

(Scheme 5; ESI, Table S2†). The tripartite structure 18 failed to

inhibit BACE1 at concentrations up to 10 μ^M. Pairwise compari-

son of the log EC₅₀values for the tripartite structures 19 and 20

and the free pharmacophores 17, 50 and 51 by one-way ANOVA

and Tukey’s post-test showed that all ﬁve differed signiﬁcantly

from each other (ESI, Fig. S1 and Table S3†). In contrast to 18,

the tripartite structures 19 and 20, which resemble structure 17

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inhibitor. The ability of the membrane anchor to bind to lipid

rafts strongly depends on its structure. This raftophilicity deter-

mines the concentration of the tripartite structure at the site of

APP cleavage and therefore the potency of our compounds. Our

observations indicate that a variation of the theoretical linker

length from 26 to 96 Å has only a small impact on the inhibitory

activity of the tripartite structure. This observation is tentatively

explained by a spacer conformation which is not fully elongated

in an aqueous environment. It is a further advantage of our

concept that tripartite structures are relatively tolerant to struc-

tural changes in the spacer.

Experimental

General

The spacer building blocks 4a and 4b, and Fmoc-statine were

purchased from Polypeptide, France. The spacer building blocks

4c–g were purchased from Iris Biotech, Germany. The other

amino acids and activation agents were bought from Novabio-

chem/Merck. All reactions were carried out using dry solvents in

oven-dried glassware under an argon atmosphere. Solvents were

dried using a solvent puriﬁcation system (MBraun-SPS). Other

chemicals were used as received from commercial sources. Flash

chromatography was carried out using silica gel from Acros

Organics (0.035–0.070 mm) and a Sepacore system (Büchi). The

tripartite structures were assembled by automated solid-phase

peptide synthesis on an ABI 433A peptide synthesiser with a

UV detector (Fmoc-strategy, PAL-PEG-PS resin, HATU or

HBTU activation). ATR FT-IR spectra were recorded on a

Thermo Nicolet Avatar 360. NMR spectra were recorded on a

Bruker Avance II 300, on a Bruker DRX 500 and an Avance III

600; δ in ppm with the non-deuterated solvent as an internal

standard,⁴⁵J in Hz. Mass spectra were measured on a Bruker

Esquire LC Electrospray MS. The most abundant mass is given

as theoretical mass. HRMS was measured on an LTQ ORBI-

TRAP XL instrument (ThermoScientiﬁc) with a resolution of

60 000 and a mass accuracy within 3 ppm. Analytical HPLC

was carried out on an Agilent Model 1100 with G1315B

UV-DAD (detection at 215, 260 and 560 nm), G1321A ﬂuor-

escence and an evaporative light scattering detector (ELS 1000,

Polymer Laboratories). Column: Vydac 208TP104 (reverse-

phase C₈, 4.6 × 250 mm). Flow rate: 1.0 mL min⁻¹. Eluent A:

H₂O + 0.1% CF₃COOH, eluent B: MeCN + 0.1% CF₃COOH.

Gradient from 20% to 90% B in 35 min. Preparative HPLC was

carried out using a Varian PrepStar system, with a Varian ProStar

Model 320 UV and an evaporative light scattering detector (ELS

1000, Polymer Laboratories) connected via a Sunchrom Quick-

Split splitter. Column: Vydac 208TP1050 (reverse-phase C₈, 50 ×

250 mm). Flow rate: 55 mL min⁻¹. Eluent A: H₂O + 0.1%

CF₃COOH, eluent B: MeCN + 0.1% CF₃COOH. Gradient stated

for the individual compounds.

Scheme 5 Synthesis of the building blocks 44, 49 and 50. Reagents

and conditions: (a) 1.9 equiv. Fmoc-Glu-OtBu (43), 1.9 equiv. HATU,

4.3 equiv. EtN(i-Pr)₂, CH₂Cl₂, rt, 18 h; (b) CF₃COOH, CH₂Cl₂(1 : 1),

rt, 90 min, 57% (two steps); (c) 1.4 equiv. 45 or 2.3 equiv. 46, 3.0 equiv.

NaBH(OAc)₃, 1.5 equiv. EtN(i-Pr)₂, 1,2-C₂H₄Cl₂, rt, 18–19 h, 70% for

47, 73% for 48; (d) CF₃COOH, CH₂Cl₂(1 : 1), rt, 90 min, 78%; (e) 10.0

wt-equiv. silica gel, toluene, 111 °C, 6 h, 86%; (f) piperidine, DMF

(1 : 5), rt, 3.5 h, 75%.

more closely, were highly active. The potency of 20, the most

active tripartite structure within this series, exceeded that of

inhibitor 17 by almost two orders of magnitude. This outcome

emphasises that the potency of intrinsically cell-permeable

BACE1 inhibitors can be enhanced considerably by membrane

anchoring. The tripartite structure 20 is signiﬁcantly more active

than 19, which differs from the former only by the presence of

an amino group in the linkage to the pharmacophore. Obviously,

decreased polarity in the region of the hydrophobic S₃binding

pocket results in increased activity.

Conclusions

The present results provide strong evidence for our tripartite

structure concept.²⁴We have demonstrated that the cellular

activity of small-molecule BACE1 inhibitors can be signiﬁcantly

increased by membrane targeting via a spacer with a raftophilic

membrane anchor. A careful selection of the coupling strategy is

crucial to avoid alteration of essential pharmacophoric groups of

the inhibitor which may lead to a loss of inhibitory activity. The

tripartite structure concept leads to an enrichment of the mem-

brane-bound inhibitor in the lipid rafts which may reduce

unwanted off-target side effects caused by the free BACE1

Fmoc-Asp(ODhc)-OH (3a). For synthesis and spectroscopic

data, see ref. 26.

Fmoc-Asp(OChol)-OH (3b). Cholesterol (1.29 g, 3.33 mmol)

and a catalytic amount of DMAP were added to a solution of

Fmoc-Asp-OtBu (1) (1.99 g, 4.83 mmol) and EDC·HCl

(0.938 g, 4.89 mmol) in CH₂Cl₂(60 mL). After stirring for 52 h

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at room temperature, the mixture was partitioned between 2%

aqueous citric acid and CH₂Cl₂, washed with 2% citric acid,

H₂O and brine. The organic layer was dried over Na₂SO₄and

evaporated to yield a colourless solid, which was dissolved in

CH₂Cl₂(30 mL). CF₃COOH (30 mL) was added at 0 °C, the

mixture was stirred at room temperature for 90 min, diluted with

CH₂Cl₂and washed four times with water and once with brine.

After drying over Na₂SO₄and removal of the solvent, the

residue was puriﬁed by ﬂash chromatography (SiO₂; pentane/

ethyl acetate [2 : 1] + 0.5% AcOH), affording 3b as a colourless

solid, yield: 1.46 g (61%), m.p. 157–158 °C. IR (ATR): ν =

2932, 2867, 2850, 1725, 1703, 1653, 1558, 1515, 1447, 1376,

1330, 1273, 1218, 1084, 1029, 1008, 957, 849, 801, 757,

14.28 (CH₃), 22.84 (CH₂), 26.00 (CH₂), 28.61 (CH₂), 29.37

(CH₂), 29.51 (CH₂), 29.67 (CH₂), 29.73 (CH₂), 29.80 (3 CH₂),

29.84 (CH₂), 32.06 (CH₂), 36.54 (CH₂), 47.20 (CH), 50.31

(CH), 65.81 (CH₂), 67.60 (CH₂), 120.14 (2 CH), 125.26 (CH),

125.30 (CH), 127.23 (2 CH), 127.88 (2 CH), 141.43 (2 C),

143.76 (C), 143.89 (C), 156.26 (CvO), 171.43 (CvO), 175.69

(CvO). ESI-MS: m/z = 552.3 [M + H]⁺, 569.3 [M + NH₄]⁺.

Fmoc-Asp(O-C₁₆H₃₃)-OH (3d). A solution of Fmoc-Asp-

OtBu (1) (1.00 g, 2.43 mmol), MSNT (0.721 g, 2.43 mmol) and

MeIm (0.204 g, 2.43 mmol) in CH₂Cl₂(60 mL) was stirred for

10 min and palmityl alcohol (0.651 g, 2.68 mmol) was added

subsequently. After stirring for 4 h at room temperature, the

mixture was partitioned between 2% aqueous citric acid and

CH₂Cl₂, washed with 2% citric acid, H₂O and brine. After

drying over Na₂SO₄and removal of the solvent, the residue was

puriﬁed by ﬂash chromatography (SiO₂; petroleum ether/diethyl

ether, 2 : 1), affording the protected ester as a colourless solid,

yield: 1.27 g (75%). This product (0.904 g, 1.42 mmol) was dis-

solved in CH₂Cl₂(15 mL). CF₃COOH (10 mL) was added at

0 °C, the mixture stirred at room temperature for 90 min, diluted

with CH₂Cl₂and washed four times with water and once with

brine. After drying over Na₂SO₄and removal of the solvent, the

residue was puriﬁed by ﬂash chromatography (SiO₂; petroleum

ether/diethyl ether [2 : 1] + 0.5% AcOH), affording 3d as a col-

ourless solid, yield: 0.702 g (85%). IR (ATR): ν = 3375, 3065,

2917, 2848, 2057, 1757, 1678, 1535, 1450, 1377, 1358, 1254,

735 cm⁻^{1 1}H NMR (500 MHz, CDCl₃): δ = 0.67 (s, 3 H),

.

0.85–1.62 (m, 33 H), 1.79–1.87 (m, 3 H), 1.94–2.02 (m, 2 H),

2.32 (d, J = 7.9 Hz, 2 H), 2.86 (dd, J = 17.3, 4.7 Hz, 1 H), 3.06

(dd, J = 17.6, 4.1 Hz, 1 H), 4.24 (t, J = 7.3 Hz, 1 H), 4.31–4.39

(m, 1 H), 4.40–4.47 (m, 1 H), 4.57–4.72 (m, 2 H), 5.37 (d, J =

3.8 Hz, 1 H), 5.87 (d, J = 8.2 Hz, 1 H), 7.31 (t, J = 7.6 Hz, 2 H),

7.40 (t, J = 7.6 Hz, 2 H), 7.58–7.61 (m, 2 H), 7.76 (d, J =

7.6 Hz, 2 H). ¹³C NMR and DEPT (125 MHz, CDCl₃): δ =

11.99 (CH₃), 18.86 (CH₃), 19.42 (CH₃), 21.16 (CH₂), 22.71

(CH₃), 22.97 (CH₃), 23.99 (CH₂), 24.41 (CH₂), 27.79 (CH₂),

28.16 (CH), 28.37 (CH₂), 31.95 (CH), 32.02 (CH₂), 35.94 (CH),

36.32 (CH₂), 36.69 (C), 36.88 (CH₂), 37.03 (CH₂), 38.08 (CH₂),

39.65 (CH₂), 39.83 (CH₂), 42.44 (C), 47.20 (CH), 49.10 (CH),

50.34 (CH), 56.28 (CH), 56.80 (CH), 67.55 (CH₂), 75.57 (CH),

120.15 (2 CH), 123.13 (CH), 125.25 (CH), 125.29 (CH), 127.24

(2 CH), 127.90 (2 CH), 139.40 (C), 141.44 (2 C), 143.78 (C),

143.92 (C), 156.24 (CvO), 170.80 (CvO), 175.67 (CvO).

ESI-MS: m/z = 724.5 [M + H]⁺, 741.7 [M + NH₄]⁺.

1193, 1166, 1090, 1043, 988, 861, 736 cm⁻^{1 1}H NMR

.

(500 MHz, CDCl₃): δ = 0.88 (t, J = 6.9 Hz, 3 H), 1.20–1.35 (m,

26 H), 1.59–1.65 (m, 2 H), 2.89 (dd, J = 17.4, 4.6 Hz, 1 H),

3.10 (dd, J = 17.4, 4.1 Hz, 1 H), 4.12 (t, J = 6.7 Hz, 2 H), 4.24

(t, J = 7.2 Hz, 1 H), 4.33–4.37 (m, 1 H), 4.41–4.46 (m, 1 H),

4.66–4.72 (m, 1 H), 5.84 (br d, J = 8.5 Hz, 1 H), 7.31 (t, J =

7.3 Hz, 2 H), 7.40 (t, J = 7.4 Hz, 2 H), 7.56–7.61 (m, 2 H), 7.75

(d, J = 7.5 Hz, 2 H). ¹³C NMR and DEPT (125 MHz, CDCl₃): δ

= 14.29 (CH₃), 22.85 (CH₂), 26.00 (CH₂), 28.61 (CH₂), 29.39

(CH₂), 29.52 (CH₂), 29.67 (CH₂), 29.74 (CH₂), 29.81 (CH₂),

29.84 (5 CH₂), 32.08 (CH₂), 36.52 (CH₂), 47.21 (CH), 50.28

(CH), 65.82 (CH₂), 67.58 (CH₂), 120.15 (2 CH), 125.25 (CH),

125.28 (CH), 127.24 (2 CH), 127.90 (2 CH), 141.44 (2 C),

143.76 (C), 143.90 (C), 156.25 (CvO), 171.46 (CvO), 175.33

(CvO). ESI-MS: m/z = 580.3 [M + H]⁺, 597.3 [M + NH₄]⁺.

Fmoc-Asp(O-C₁₄H₂₉)-OH (3c). A solution of Fmoc-Asp-

OtBu (1) (5.56 g, 13.5 mmol), MSNT (4.00 g, 13.5 mmol) and

MeIm (1.11 g, 13.5 mmol) in CH₂Cl₂(60 mL) was stirred for

10 min and myristyl alcohol (2.63 g, 12.3 mmol) was added sub-

sequently. After stirring for 4 h at room temperature, the mixture

was partitioned between 2% aqueous citric acid and CH₂Cl₂,

washed with 2% citric acid, H₂O and brine. After drying over

Na₂SO₄and removal of the solvent, the residue was puriﬁed by

ﬂash chromatography (SiO₂; petroleum ether/diethyl ether, 2 : 1),

affording the protected ester as a colourless solid, yield: 5.56 g

(76%). This product (5.00 g, 8.23 mmol) was dissolved in

CH₂Cl₂(15 mL). CF₃COOH (10 mL) was added at 0 °C, the

mixture stirred at room temperature for 90 min, diluted with

CH₂Cl₂and washed four times with water and once with brine.

After drying over Na₂SO₄and removal of the solvent, the

residue was puriﬁed by ﬂash chromatography (SiO₂; petroleum

ether/diethyl ether [2 : 1] + 0.5% AcOH), affording 3c as a col-

ourless solid, yield: 3.99 g (88%). IR (ATR): ν = 3355, 3247,

2920, 2851, 2057, 1724, 1652, 1543, 1470, 1457, 1404, 1372,

Fmoc-Asp(O-C₁₈H₃₇)-OH (3e). A solution of Fmoc-Asp-

OtBu (1) (0.503 g, 1.22 mmol), MSNT (0.363 g, 1.21 mmol)

and MeIm (99 mg, 1.22 mmol) in CH₂Cl₂(20 mL) was stirred

for 10 min and stearyl alcohol (301 mg, 1.11 mmol) was added

subsequently. After stirring for 14 h at room temperature, the

mixture was partitioned between 2% aqueous citric acid and

CH₂Cl₂, washed with 2% citric acid, H₂O and brine. The

organic layer was dried over Na₂SO₄and evaporated to yield a

colourless viscous oil, which was dissolved in CH₂Cl₂(5 mL).

After addition of CF₃COOH (6.25 mL) at 0 °C, the mixture was

stirred at room temperature for 90 min, diluted with CH₂Cl₂and

washed four times with water and once with brine. After drying

over Na₂SO₄and removal of the solvent, the residue was

puriﬁed by ﬂash chromatography (SiO₂; petroleum ether/ethyl

acetate [2 : 1] + 0.5% AcOH), affording 3e as a colourless solid,

yield: 0.568 g (76%), m.p. 97–99 °C. IR (ATR): ν = 3377, 3064,

1280, 1237, 1184, 1083, 995, 832, 809, 770, 735 cm⁻^{1 1}H

.

NMR (500 MHz, CDCl₃): δ = 0.88 (t, J = 6.9 Hz, 3 H),

1.22–1.31 (m, 22 H), 1.59–1.65 (m, 2 H), 2.89 (d, J = 15.5 Hz,

1 H), 3.10 (d, J = 15.5 Hz, 1 H), 4.12 (t, J = 6.4 Hz, 2 H), 4.24

(t, J = 6.6 Hz, 1 H), 4.33–4.37 (m, 1 H), 4.42–4.46 (m, 1 H),

4.70 (br s, 1 H), 5.87 (br d, J = 7.7 Hz, 1 H), 7.31 (t, J = 7.3 Hz,

2 H), 7.40 (t, J = 7.4 Hz, 2 H), 7.57–7.61 (m, 2 H), 7.76 (d, J =

7.5 Hz, 2 H). ¹³C NMR and DEPT (125 MHz, CDCl₃): δ =

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2916, 2849, 1757, 1734, 1693, 1678, 1533, 1472, 1450, 1377,

1358, 1322, 1253, 1195, 1166, 1089, 1043, 990, 860, 736 cm⁻¹

organic layer was dried over Na₂SO₄and evaporated to yield a

colourless solid, which was dissolved in CH₂Cl₂(60 mL).

CF₃COOH (60 mL) was added at 0 °C, the mixture stirred at

room temperature for 90 min, diluted with CH₂Cl₂and washed

four times with water and once with brine. After drying over

Na₂SO₄and removal of the solvent, the residue was puriﬁed by

ﬂash chromatography (SiO₂; petroleum ether/ethyl acetate [2 : 1]

+ 0.5% AcOH), affording 3g as a colourless solid, yield: 2.28 g

(92%), m.p. 98–100 °C. IR (ATR): ν = 3387, 3066, 2916, 2849,

1755, 1732, 1712, 1539, 1515, 1472, 1450, 1380, 1359, 1320,

1252, 1206, 1091, 1041, 989, 965, 935, 860, 775, 756, 737,

718 cm⁻¹. ¹H NMR (500 MHz, CDCl₃): δ = 0.88 (t, J = 6.8 Hz,

3 H), 1.19–1.30 (m, 38 H), 1.57–1.68 (m, 2 H), 2.89 (dd, J =

17.4, 4.5 Hz, 1 H), 3.09 (dd, J = 17.4, 4.2 Hz, 1 H), 4.11 (t, J =

6.7 Hz, 2 H), 4.23 (t, J = 7.2 Hz, 1 H), 4.31–4.48 (m, 2 H),

4.63–4.72 (m, 1 H), 5.89 (d, J = 8.5 Hz, 1 H), 7.31 (t, J =

7.3 Hz, 2 H), 7.40 (t, J = 7.4 Hz, 2 H), 7.55–7.63 (m, 2 H), 7.76

(d, J = 7.5 Hz, 2 H). ¹³C NMR and DEPT (125 MHz, CDCl₃): δ

= 14.28 (CH₃), 22.84 (CH₂), 26.00 (CH₂), 28.60 (CH₂), 29.39

(CH₂), 29.51 (CH₂), 29.68 (CH₂), 29.75 (CH₂), 29.81 (CH₂),

29.86 (11 CH₂), 32.07 (CH₂), 36.52 (CH₂), 47.20 (CH), 50.30

(CH), 65.79 (CH₂), 67.57 (CH₂), 120.14 (2 CH), 125.25 (CH),

125.29 (CH), 127.24 (2 CH), 127.89 (2 CH), 141.43 (2 C),

143.77 (C), 143.90 (C), 156.26 (CvO), 171.42 (CvO), 175.61

(CvO). ESI-MS: m/z = 664.4 [M + H]⁺, 681.5 [M + NH₄]⁺.

.

¹H NMR (500 MHz, CDCl₃): δ = 0.88 (t, J = 6.9 Hz, 3 H),

1.17–1.34 (m, 30 H), 1.57–1.69 (m, 2 H), 2.89 (dd, J = 17.4,

4.5 Hz, 1 H), 3.09 (dd, J = 17.3, 3.9 Hz, 1 H), 4.12 (t, J = 6.7

Hz, 2 H), 4.23 (t, J = 7.2 Hz, 1 H), 4.32–4.40 (m, 1 H),

4.39–4.49 (m, 1 H), 4.64–4.75 (m, 1 H), 5.86 (d, J = 8.3 Hz, 1

H), 7.31 (t, J = 7.4 Hz, 2 H), 7.40 (t, J = 7.4 Hz, 2 H), 7.59 (d,

J = 5.8 Hz, 2 H), 7.76 (d, J = 7.5 Hz, 2 H). ¹³C NMR and DEPT

(125 MHz, CDCl₃): δ = 14.28 (CH₃), 22.84 (CH₂), 26.00 (CH₂),

28.60 (CH₂), 29.39 (CH₂), 29.51 (CH₂), 29.67 (CH₂), 29.74

(CH₂), 29.81 (CH₂), 29.85 (7 CH₂), 32.07 (CH₂), 36.55 (CH₂),

47.21 (CH), 50.30 (CH), 65.79 (CH₂), 67.55 (CH₂), 120.14

(2 CH), 125.25 (CH), 125.29 (CH), 127.24 (2 CH), 127.90 (2

CH), 141.44 (2 C), 143.78 (C), 143.90 (C), 156.26 (CvO),

171.51 (CvO), 174.46 (CvO). ESI-MS: m/z = 608.3 [M + H]⁺,

625.4 [M + NH₄]⁺.

Fmoc-Asp(O-C₂₀H₄₁)-OH (3f). A solution of Fmoc-Asp-

OtBu (1) (1.51 g, 3.68 mmol), MSNT (1.09 g, 3.67 mmol) and

MeIm (0.311 g, 3.67 mmol) in CH₂Cl₂(60 mL) was stirred for

10 min and arachidyl alcohol (1.00 g, 3.36 mmol) was added

subsequently. After stirring for 17 h at room temperature, the

mixture was partitioned between 2% aqueous citric acid and

CH₂Cl₂, washed with 2% citric acid, H₂O and brine. The

organic layer was dried over Na₂SO₄and evaporated to yield a

colourless viscous oil, which was dissolved in CH₂Cl₂(60 mL).

CF₃COOH (60 mL) was added at 0 °C, the mixture stirred at

room temperature for 90 min, diluted with CH₂Cl₂and washed

four times with water and once with brine. After drying over

Na₂SO₄and removal of the solvent, the residue was puriﬁed by

ﬂash chromatography (SiO₂; petroleum ether/ethyl acetate [2 : 1]

+ 0.5% AcOH), affording 3f as a colourless solid, yield: 1.45 g

(68%), m.p. 97–99 °C. IR (ATR): ν = 3387, 3066, 2916, 2849,

1755, 1734, 1713, 1693, 1512, 1472, 1450, 1378, 1359, 1320,

1252, 1193, 1166, 1090, 1042, 992, 860, 775, 755, 736,

720 cm⁻¹. ¹H NMR (500 MHz, CDCl₃): δ = 0.88 (t, J = 6.9 Hz,

3 H), 1.19–1.35 (m, 34 H), 1.60–1.66 (m, 2 H), 2.89 (dd, J =

17.4, 4.7 Hz, 1 H), 3.09 (dd, J = 17.4, 4.2 Hz, 1 H), 4.12 (t, J =

6.7 Hz, 2 H), 4.24 (t, J = 7.1 Hz, 1 H), 4.36 (s, 1 H), 4.41–4.48

(m, 1 H), 4.67–4.75 (m, 1 H), 5.85 (d, J = 8.4 Hz, 1 H), 7.31 (t,

J = 7.2 Hz, 2 H), 7.40 (t, J = 7.4 Hz, 2 H), 7.59 (d, J = 5.4 Hz,

2 H), 7.76 (d, J = 7.5 Hz, 2 H). ¹³C NMR and DEPT (125 MHz,

CDCl₃): δ = 14.28 (CH₃), 22.84 (CH₂), 25.99 (CH₂), 28.60

(CH₂), 29.38 (CH₂), 29.51 (CH₂), 29.67 (CH₂), 29.74 (CH₂),

29.81 (CH₂), 29.85 (9 CH₂), 32.07 (CH₂), 36.52 (CH₂), 47.21

(CH), 50.25 (CH), 65.82 (CH₂), 67.57 (CH₂), 120.15 (2 CH),

125.24 (CH), 125.28 (CH), 127.24 (2 CH), 127.90 (2 CH),

141.44 (2 C), 143.77 (C), 143.89 (C), 156.24 (CvO), 171.49

(CvO), 174.87 (CvO). ESI-MS: m/z = 636.6 [M + H]⁺, 653.5

[M + NH₄]⁺.

Fmoc-Asp(O-C₂₄H₄₉)-OH (3h). A solution of Fmoc-Asp-

OtBu (1) (0.127 g, 0.314 mmol), MSNT (91.8 mg, 0.31 mmol)

and MeIm (25.3 mg, 0.312 mmol) in CH₂Cl₂(25 mL) was

stirred for 10 min and lignoceryl alcohol (0.100 g, 0.284 mmol)

was added subsequently. After stirring for 24 h at room tempera-

ture, the mixture was partitioned between 2% aqueous citric acid

and CH₂Cl₂, washed with 2% citric acid, H₂O and brine. The

organic layer was dried over Na₂SO₄and evaporated to yield a

colourless solid, which was dissolved in CH₂Cl₂(5 mL).

CF₃COOH (6.25 mL) was added at 0 °C, the mixture stirred at

room temperature for 90 min, diluted with CH₂Cl₂and washed

four times with water and once with brine. After drying over

Na₂SO₄and removal of the solvent, the residue was puriﬁed by

ﬂash chromatography (SiO₂; petroleum ether/ethyl acetate [2 : 1]

+ 0.5% AcOH), affording 3h as a colourless solid, yield: 0.137 g

(70%), m.p. 97–99 °C. IR (ATR): ν = 3315, 2916, 2848, 1770,

1735, 1690, 1652, 1636, 1535, 1464, 1449, 1412, 1398, 1369,

1340, 1281, 1231, 1176, 1105, 1087, 1049, 1015, 921, 779, 756,

1

735, 637 cm⁻¹. H NMR (500 MHz, CDCl₃): δ = 0.88 (t, J =

7.0 Hz, 3 H), 1.23–1.36 (m, 42 H), 1.59–1.69 (m, 2 H), 2.88

(dd, J = 17.4, 4.7 Hz, 1 H), 3.08 (dd, J = 17.4, 4.3 Hz, 1 H),

4.12 (t, J = 6.7 Hz, 2 H), 4.23 (t, J = 7.1 Hz, 1 H), 4.34–4.40

(m, 1 H), 4.42–4.49 (m, 1 H), 4.69–4.72 (m, 1 H), 5.86 (d, J =

8.5 Hz, 1 H), 7.31 (t, J = 7.3 Hz, 2 H), 7.40 (t, J = 7.5 Hz, 2 H),

7.60 (d, J = 5.2 Hz, 2 H), 7.76 (d, J = 7.5 Hz, 2 H). ¹³C NMR

and DEPT (125 MHz, CDCl₃): δ = 14.28 (CH₃), 22.84 (CH₂),

26.00 (CH₂), 28.61 (CH₂), 29.39 (CH₂), 29.51 (CH₂), 29.68

(CH₂), 29.75 (CH₂), 29.81 (CH₂), 29.86 (13 CH₂), 32.07 (CH₂),

36.52 (CH₂), 47.21 (CH), 50.36 (CH), 65.82 (CH₂), 67.57

(CH₂), 120.15 (2 CH), 125.25 (CH), 125.28 (CH), 127.24 (2

CH), 127.90 (2 CH), 141.44 (2 C), 143.77 (C), 143.89 (C),

156.25 (CvO), 171.49 (CvO), 174.91 (CvO). ESI-MS: m/z =

692.6 [M + H]⁺, 709.7 [M + NH₄]⁺.

Fmoc-Asp(O-C₂₂H₄₅)-OH (3g). A solution of Fmoc-Asp-

OtBu (1) (1.68 g, 4.09 mmol), MSNT (1.21 g, 4.08 mmol) and

MeIm (0.335 g, 4.09 mmol) in CH₂Cl₂(60 mL) was stirred for

10 min and behenyl alcohol (1.22 g, 3.72 mmol) was added sub-

sequently. After stirring for 24 h at room temperature, the

mixture was partitioned between 2% aqueous citric acid and

CH₂Cl₂, washed with 2% citric acid, H₂O and brine. The

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Fmoc-Asp(O-C₂₆H₅₃)-OH (3i). A solution of Fmoc-Asp-OtBu

(1) (0.118 g, 0.288 mmol), MSNT (85.6 mg, 0.289 mmol) and

MeIm (24.2 mg, 0.295 mmol) in CH₂Cl₂(25 mL) was stirred for

10 min and ceryl alcohol (0.100 g, 0.261 mmol) was added sub-

sequently. After stirring for 25 h at room temperature, the

mixture was partitioned between 2% aqueous citric acid and

CH₂Cl₂, washed with 2% citric acid, H₂O and brine. The

organic layer was dried over Na₂SO₄and evaporated to yield a

colourless solid, which was dissolved in CH₂Cl₂(5 mL).

CF₃COOH (6.25 mL) was added at 0 °C, the mixture stirred at

room temperature for 90 min, diluted with CH₂Cl₂and washed

four times with water and once with brine. After drying over

Na₂SO₄and removal of the solvent, the residue was puriﬁed by

ﬂash chromatography (SiO₂; petroleum ether/ethyl acetate [2 : 1]

+ 0.5% AcOH), affording 3i as a colourless solid, yield: 0.151 g

(80%), m.p. 99–101 °C. IR (ATR): ν = 3379, 2916, 2849, 2057,

1758, 1734, 1535, 1472, 1450, 1377, 1359, 1322, 1253, 1203,

7.4 Hz, 2 H), 7.40 (t, J = 7.4 Hz, 2 H), 7.60 (d, J = 5.7 Hz, 2 H),

7.76 (d, J = 7.5 Hz, 2 H).¹³C NMR and DEPT (125 MHz,

CDCl₃): δ = 14.28 (CH₃), 22.84 (CH₂), 25.99 (CH₂), 27.34

(CH₂), 27.36 (CH₂), 28.60 (CH₂), 29.36 (2 CH₂), 29.47 (2

CH₂), 29.56 (CH₂), 29.68 (CH₂), 29.88 (CH₂), 29.91 (CH₂),

32.05 (CH₂), 36.51 (CH₂), 47.21 (CH), 50.25 (CH), 65.82

(CH₂), 67.58 (CH₂), 120.16 (2 CH), 125.28 (CH), 125.48 (CH),

127.24 (2 CH), 127.91 (2 CH), 129.91 (CH), 130.14 (CH),

141.44 (2 C), 143.77 (C), 143.89 (C), 156.24 (CvO), 171.52

(CvO), 174.81 (CvO). ESI-MS: m/z = 606.3 [M + H]⁺.

i-Bu-Lys(Cbz)-OtBu. Acetic acid (77 μL, 1.4 mmol), a solu-

tion of Lys(Cbz)-OtBu·HCl (21) (0.209 g, 0.561 mmol) and

EtN(i-Pr)₂(64 μL, 0.37 mmol) in methanol (4 mL), and ﬁnally

isobutyraldehyde (58 μL, 0.64 mmol) were added one after the

other to a solution of NaBH₃CN (40.3 mg, 0.642 mmol) in

methanol (10 mL). The mixture was stirred at room temperature

for 2 h. The major part of methanol was removed and the

mixture was partitioned between ethyl acetate and K₂CO₃(half-

sat. aqueous). The organic layer was washed with brine and

dried over Na₂SO₄. Removal of the solvent and ﬂash chromato-

graphy (SiO₂; petroleum ether/ethyl acetate, 2 : 3) afforded i-Bu-

Lys(Cbz)-OtBu as a colourless oil, yield: 0.194 g (92%). IR

(ATR): ν = 3329, 2948, 2933, 2867, 1771, 1723, 1709, 1653,

1531, 1452, 1395, 1367, 1338, 1243, 1150, 1081, 1048, 1027,

845, 777, 738, 696, 651 cm⁻¹. ¹H NMR (500 MHz, CDCl₃): δ =

0.89 (d, J = 6.6 Hz, 6 H), 1.36–1.42 (m, 1 H), 1.46 (s, 9 H),

1.48–1.61 (m, 5 H), 1.64–1.71 (m, 1 H), 2.26 (dd, J = 11.1,

6.1 Hz, 1 H), 2.35 (dd, J = 11.1, 7.4 Hz, 1 H), 3.02 (t, J = 6.7

Hz, 1 H), 3.19 (q, J = 6.6 Hz, 2 H), 4.82 (br s, 1 H), 5.08 (s,

2 H), 7.29–7.37 (m, 5 H). ¹³C NMR and DEPT (125 MHz,

CDCl₃): δ = 20.61 (CH₃), 20.96 (CH₃), 23.15 (CH₂), 28.36 (3

CH₃), 28.81 (CH), 29.82 (CH₂), 33.28 (CH₂), 41.01 (CH₂),

56.32 (CH₂), 62.41 (CH), 66.70 (CH₂), 81.03 (C), 128.20 (2

CH), 128.24 (CH), 128.63 (2 CH), 136.76 (C), 156.48 (CvO),

175.14 (CvO). ESI-MS: m/z = 393.4 [M + H]⁺.

1168, 1090, 1043, 989, 937, 860, 776, 755, 736, 718, 650 cm⁻¹

.

¹H NMR (500 MHz, CDCl₃): δ = 0.88 (t, J = 6.9 Hz, 3 H),

1.20–1.36 (m, 46 H), 1.59–1.69 (m, 2 H), 2.89 (dd, J = 17.4,

4.7 Hz, 1 H), 3.09 (dd, J = 17.4, 4.2 Hz, 1 H), 4.10–4.15 (m, 2

H), 4.24 (t, J = 7.1 Hz, 1 H), 4.34–4.38 (m, 1 H), 4.43–4.46 (m,

1 H), 4.68–4.71 (m, 1 H), 5.86 (d, J = 8.5 Hz, 1 H), 7.31 (t, J =

7.3 Hz, 2 H), 7.40 (t, J = 7.5 Hz, 2 H), 7.59 (d, J = 6.2 Hz, 2 H),

7.76 (d, J = 7.6 Hz, 2 H). ¹³C NMR and DEPT (125 MHz,

CDCl₃): δ = 14.28 (CH₃), 22.84 (CH₂), 25.99 (CH₂), 28.60

(CH₂), 29.39 (CH₂), 29.51 (CH₂), 29.75 (CH₂), 29.81 (CH₂),

29.85 (16 CH₂), 32.07 (CH₂), 36.52 (CH₂), 47.21 (CH), 50.23

(CH), 65.83 (CH₂), 67.57 (CH₂), 120.15 (2 CH), 125.24 (CH),

125.28 (CH), 127.24 (2 CH), 127.91 (2 CH), 141.44 (2 C),

143.77 (C), 143.89 (C), 156.24 (CvO), 171.54 (CvO), 174.31

(CvO). ESI-MS: m/z = 720.7 [M + H]⁺, 737.7 [M + NH₄]⁺.

Fmoc-Asp(O-Oleyl)-OH (3j). A solution of Fmoc-Asp-OtBu

(1) (5.43 g, 13.2 mmol), MSNT (3.91 g, 13.2 mmol) and MeIm

(1.08 g, 13.2 mmol) in CH₂Cl₂(15 mL) was stirred for 10 min

and oleyl alcohol (2.50 g, 12.0 mmol) was added subsequently.

After stirring for 4 h at room temperature, the mixture was parti-

tioned between 2% aqueous citric acid and CH₂Cl₂, washed with

2% citric acid, H₂O and brine. After drying over Na₂SO₄and

removal of the solvent, the residue was puriﬁed by ﬂash chrom-

atography (SiO₂; petroleum ether/diethyl ether, 2 : 1), affording

the protected ester as a colourless solid, yield: 6.20 g (78%).

This product (6.00 g, 9.07 mmol) was dissolved in CH₂Cl₂

(25 mL). CF₃COOH (15 mL) was added at 0 °C, the mixture

stirred at room temperature for 90 min, diluted with CH₂Cl₂and

washed four times with water and once with brine. After drying

over Na₂SO₄and removal of the solvent, the residue was

puriﬁed by ﬂash chromatography (SiO₂; petroleum ether/diethyl

ether [2 : 1] + 0.5% AcOH), affording 3j as a colourless solid,

yield: 4.56 g (83%). IR (ATR): ν = 3354, 3246, 3064, 3005,

2921, 2852, 2056, 1724, 1703, 1652, 1543, 1450, 1404, 1372,

1280, 1236, 1184, 1083, 994, 833, 810, 770, 734, 620 cm⁻¹. ¹H

NMR (500 MHz, CDCl₃): δ = 0.88 (t, J = 6.9 Hz, 3 H),

1.26–1.37 (m, 22 H), 1.62–1.65 (m, 2 H), 1.99–2.02 (m, 4 H),

2.89 (dd, J = 4.2, 17.5 Hz, 1 H), 3.10 (dd, J = 3.8, 17.4 Hz,

1 H), 4.13 (t, J = 6.6 Hz, 2 H), 4.24 (t, J = 7.0 Hz, 1 H),

4.34–4.38 (m, 1 H), 4.43–4.46 (m, 1 H), 4.69–4.71 (m, 1 H),

5.32–5.36 (m, 2 H), 5.85 (br d, J = 8.3 Hz, 1 H), 7.32 (t, J =

Benzyl 4-iodophenylacetate. Benzyl bromide (58 μL,

0.49 mmol) was added dropwise to a mixture of 4-iodophenyl-

acetic acid (0.101 g, 0.385 mmol) and Cs₂CO₃(0.162 g,

0.497 mmol) in DMF (10 mL). The reaction mixture was stirred

at room temperature for 1 h and then partitioned between ethyl

acetate and H₂O. The organic layer was washed twice with H₂O,

once with brine and dried over Na₂SO₄. Removal of the solvent

and ﬂash chromatography (SiO₂; petroleum ether/ethyl acetate,

50 : 1 to 3 : 1) afforded benzyl 4-iodophenylacetate as a colour-

less solid, yield: 0.124 g (92%), m.p. 49–50 °C. IR (ATR): ν =

3031, 2955, 2893, 2056, 2030, 2010, 1918, 1844, 1771, 1722,

1684, 1635, 1485, 1457, 1416, 1399, 1330, 1216, 1165, 1058,

1

1006, 974, 911, 847, 796, 754, 699 cm⁻¹. H NMR (500 MHz,

CDCl₃): δ = 3.61 (s, 2 H), 5.14 (s, 2 H), 7.03 (d, J = 8.3 Hz,

2 H), 7.30–7.38 (m, 5 H), 7.65 (d, J = 8.3 Hz, 2 H). ¹³C NMR

and DEPT (125 MHz, CDCl₃): δ = 40.93 (CH₂), 66.96 (CH₂),

92.83 (C), 128.35 (2 CH), 128.49 (CH), 128.73 (2 CH), 131.45

(2 CH), 133.61 (C), 135.75 (C), 137.79 (2 CH), 171.01 (CvO).

MS (EI): m/z (%) = 352 (28) [M⁺], 217 (84), 91 (100), 90 (20),

89 (17). Elemental analysis calcd for C₁₅H₁₃IO₂: C 51.16,

H 3.72; found: C 51.24, H 3.70.

This journal is © The Royal Society of Chemistry 2012

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Benzyl

4-benzoylthiophenylacetate. Thiobenzoic

acid

6.6 Hz, 3 H), 1.29 (s, 9 H), 1.44–1.62 (m, 5 H), 1.85–1.99 (m,

2 H), 2.95 (dd, J = 14.5, 7.4 Hz, 1 H), 3.06–3.17 (m, 3 H), 3.70

(s, 2 H), 4.23 (t, J = 7.3 Hz, 1 H), 4.86 (br s, 1 H), 5.10 (s, 2 H),

5.12 (s, 2 H), 7.29–7.40 (m, 12 H), 7.78 (d, J = 8.3 Hz, 2 H).

¹³C NMR and DEPT (125 MHz, CDCl₃): δ = 20.33 (CH₃),

20.39 (CH₃), 23.65 (CH₂), 27.78 (3 CH₃), 28.03 (CH), 29.34

(CH₂), 30.28 (CH₂), 40.71 (CH₂), 40.93 (CH₂), 53.86 (CH₂),

60.76 (CH), 66.53 (CH₂), 66.99 (CH₂), 82.02 (C), 127.77

(2 CH), 128.06 (CH), 128.08 (CH), 128.26 (2 CH), 128.43 (2

CH), 128.49 (2 CH), 128.61 (2 CH), 129.84 (2 CH), 135.40 (C),

136.62 (C), 138.61 (C), 139.12 (C), 156.36 (CvO), 169.62

(CvO), 170.34 (CvO). ESI-MS: m/z = 698.6 [M + NH₄]⁺.

23: Compound 22 (89.3 mg, 0.132 mmol) and palladium on

activated carbon (90 mg, 10 wt%) in ethyl acetate (10 mL) were

stirred under hydrogen (1 atm) at room temperature for 23 h. Fil-

tration of the reaction mixture through a short path of Celite®

and removal of the solvent afforded 23 as a colourless, viscous

oil, yield: 56.9 mg (95%). IR (ATR): ν = 3055, 2959, 2929,

2870, 2056, 2030, 1917, 1770, 1728, 1631, 1577, 1460, 1431,

1366, 1336, 1256, 1152, 1089, 1025, 992, 920, 846, 788, 723,

(0.8 mL, 6.8 mmol), EtN(i-Pr)₂(1.46 g, 2.83 mmol) and copper(I)

iodide (0.108 g, 0.567 mmol) were added to a solution of

benzyl 4-iodophenylacetate (1.99 g, 5.66 mmol) and 1,10-phe-

nanthroline (0.203 g, 1.13 mmol) in toluene (20 mL). The reac-

tion mixture was stirred at reﬂux for 18 h and then ﬁltered

through a short path of Celite®. Removal of the solvent and

ﬂash chromatography (SiO₂; petroleum ether/ethyl acetate, 20 : 1

to 2 : 1) afforded benzyl 4-benzoylthiophenylacetate as a light

brown solid, yield: 1.73 g (84%), m.p. 130–131 °C. IR (ATR):

ν = 3348, 3094, 3057, 2988, 2913, 2842, 2056, 2030, 1844,

1771, 1734, 1704, 1692, 1618, 1585, 1516, 1475, 1457, 1437,

1409, 1369, 1340, 1308, 1274, 1246, 1213, 1164, 1144, 1058,

963, 934, 856, 843, 812, 756, 722, 680, 640, 612 cm⁻^{1 1}H

.

NMR (500 MHz, CDCl₃): δ = 3.73 (s, 2 H), 5.15 (s, 2 H),

7.31–7.42 (m, 7 H), 7.46–7.52 (m, 4 H), 7.62 (t, J = 7.4 Hz,

1 H), 8.03 (d, J = 7.2 Hz, 2 H). ¹³C NMR and DEPT (125 MHz,

CDCl₃): δ = 41.24 (CH₂), 66.96 (CH₂), 126.28 (C), 127.63

(2 CH), 128.35 (2 CH), 128.46 (CH), 128.74 (2 CH), 128.90 (2

CH), 130.41 (2 CH), 133.84 (CH), 135.39 (2 CH), 135.60 (C),

135.81 (C), 136.72 (C), 171.01 (CvO), 190.27 (CvO).

ESI-MS: m/z = 742.4 [2 M + NH₄]⁺. Elemental analysis

calcd for C₂₂H₁₈O₃S: C 72.90, H 5.01, S 8.85; found: C 72.96,

H 5.10, S 8.84.

1

701, 675 cm⁻¹. H NMR (500 MHz, methanol-d₄): δ = 0.92 (d,

J = 6.6 Hz, 3 H), 0.95 (d, J = 6.6 Hz, 3 H), 1.11–1.19 (m, 1 H),

1.23–1.32 (m, 2 H), 1.38 (s, 9 H), 1.42–1.54 (m, 2 H),

1.83–2.00 (m, 2 H), 2.65–2.77 (m, 2 H), 3.00 (dd, J = 14.4, 7.4

Hz, 1 H), 3.13 (dd, J = 14.5, 7.3 Hz, 1 H), 3.63 (s, 2 H), 4.13 (t,

J = 7.3 Hz, 1 H), 7.52 (d, J = 8.3 Hz, 2 H), 7.78 (d, J = 8.3 Hz,

2 H). ¹³C NMR and DEPT (125 MHz, methanol-d₄): δ = 20.76

(CH₃), 20.82 (CH₃), 24.65 (CH₂), 28.10 (CH₂), 28.16 (3 CH₃),

29.11 (CH), 30.57 (CH₂), 40.50 (CH₂), 44.24 (CH₂), 55.86

(CH₂), 62.06 (CH), 83.31 (C), 128.53 (2 CH), 131.23 (2 CH),

139.89 (C), 143.66 (C), 171.16 2 CvO. ESI-MS: m/z = 457.3

[M + H]⁺, 479.3 [M + Na]⁺, 913.6 [2 M + H]⁺, 935.6

[2 M + Na]⁺.

Benzyl 2-(4-(chlorosulfonyl)phenyl)acetate. A solution of

benzyltrimethylammonium chloride (0.993 g, 4.97 mmol) and

trichloroisocyanuric acid (0.527 g, 2.27 mmol) in acetonitrile

(9 mL) was stirred for 30 min and added to a solution of benzyl

4-benzoylthiophenylacetate (0.500 g, 1.42 mmol) in acetonitrile

(5 mL). The reaction mixture was stirred at 0 °C for 17 min, then

diluted with ethyl acetate, washed with a dil. aqueous solution of

K₂CO₃, brine and dried over Na₂SO₄. Removal of the solvent

and instantaneous ﬂash chromatography (SiO₂; petroleum ether/

ethyl acetate, 50 : 1 to 3 : 1) afforded benzyl 2-(4-(chlorosulfo-

nyl)phenyl)acetate as white crystals, yield: 0.364 g (79%), m.p.

57–58 °C. IR (ATR): ν = 3090, 3028, 2893, 2826, 2674, 2559,

1736, 1683, 1597, 1496, 1473, 1458, 1417, 1369, 1333, 1295,

1238, 1215, 1164, 1079, 1017, 989, 930, 859, 829, 806, 741,

24: N-(FmocO)succinimide (0.239 g, 0.708 mmol) was added

to a solution of 23 (0.216 g, 0.473 mmol) in acetonitrile (4 mL),

THF (4 mL) and K₂CO₃(1 M aqueous, 4 mL). The reaction

mixture was stirred at room temperature for 17 h and then parti-

tioned between CH₂Cl₂and 2% aqueous citric acid. The organic

layer was washed with 2% citric acid, H₂O and brine and dried

over Na₂SO₄. Removal of the solvent and ﬂash chromatography

(SiO₂; petroleum ether/[CH₂Cl₂/MeOH 10 : 1] + 0.5% AcOH,

20 : 1 to 1 : 1) afforded 24 as a colourless, viscous oil, yield:

0.163 g (51%). IR (ATR): ν = 3369, 3067, 2958, 2929, 2869,

1844, 1723, 1530, 1450, 1416, 1393, 1367, 1336, 1247, 1152,

703, 676 cm⁻¹. H NMR (500 MHz, CDCl₃): δ = 3.80 (s, 2 H),

1

5.16 (s, 2 H), 7.31–7.39 (m, 5 H), 7.54 (d, J = 8.6 Hz, 2 H),

8.00 (d, J = 8.6 Hz, 2 H). ¹³C NMR and DEPT (125 MHz,

CDCl₃): δ = 41.22 (CH₂), 67.40 (CH₂), 127.41 (2 CH), 128.51

(2 CH), 128.72 (CH), 128.81 (2 CH), 130.85 (2 CH), 135.39

(C), 142.05 (C), 143.32 (C), 169.91 (CvO). ESI-MS: m/z =

322.7 [M − H]⁻.

22: EtN(i-Pr)₂(0.111 mL, 0.653 mmol) was added to a solu-

tion of iBu-Lys(Cbz)-OtBu (0.255 g, 0.65 mmol) and benzyl

2-(4-(chlorosulfonyl)phenyl)acetate (0.211 g, 0.65 mmol) in

CH₂Cl₂(20 mL). The mixture was stirred at room temperature

for 3 d and then partitioned between CH₂Cl₂and 2% aqueous

citric acid. The aqueous layer was extracted three times with

CH₂Cl₂and the combined organic layers were washed with

brine and dried over Na₂SO₄. Removal of the solvent and ﬂash

chromatography (SiO₂; pentane/ethyl acetate, 2 : 3) afforded 22

as a colourless oil, yield: 0.280 g (63%). IR (ATR): ν = 3389,

2959, 2929, 2870, 1724, 1651, 1529, 1454, 1395, 1368, 1337,

1242, 1149, 1089, 998, 845, 813, 735, 697, 677 cm⁻¹. ¹H NMR

(500 MHz, CDCl₃): δ = 0.85 (d, J = 6.6 Hz, 3 H), 0.90 (d, J =

1089, 999, 938, 845, 813, 759, 740, 651 cm⁻^{1 1}H NMR

.

(500 MHz, CDCl₃): δ = 0.92 (t, J = 7.7 Hz, 6 H), 1.02–1.11 (m,

1 H), 1.24–1.34 (m, 4 H), 1.38 (s, 9 H), 1.78–1.86 (m, 1 H),

1.89–1.96 (m, 1 H), 2.90–3.06 (m, 4 H), 3.66 (s, 2 H), 4.12 (t, J

= 7.2 Hz, 1 H), 4.20 (t, J = 6.7 Hz, 1 H), 4.41 (d, J = 6.8 Hz,

2 H), 4.84 (br t, J = 5.3 Hz, 1 H), 7.31 (t, J = 7.4 Hz, 2 H),

7.38–7.44 (m, 4 H), 7.58 (d, J = 7.4 Hz, 2 H), 7.75–7.79 (m,

4 H). ¹³C NMR and DEPT (125 MHz, CDCl₃): δ = 20.55

(CH₃), 20.57 (CH₃), 24.00 (CH₂), 27.97 (3 CH₃), 28.05 (CH),

29.42 (2 CH₂), 40.90 (CH₂), 41.24 (CH₂), 47.30 (CH), 53.73

(CH₂), 60.63 (CH), 66.95 (CH₂), 82.30 (C), 120.13 (2 CH),

125.15 (2 CH), 127.20 (2 CH), 127.70 (2 CH), 127.84 (2 CH),

130.11 (2 CH), 139.02 (C), 139.45 (C), 141.44 (2 C), 143.99

(2 C), 157.00 (CvO), 170.01 (CvO), 173.79 (CvO). ESI-MS:

m/z = 679.5 [M + H]⁺, 696.3 [M + NH₄]⁺, 701.6 [M + Na]⁺.

8228 | Org. Biomol. Chem., 2012, 10, 8216–8235

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25: EtN(i-Pr)₂(1.25 g, 9.61 mmol) and 4-toluenesulfonyl

chloride (1.22 g, 6.42 mmol) were added to a solution of i-Bu-

Lys(Cbz)-OtBu (2.52 g, 6.42 mmol) in CH₂Cl₂(60 mL). The

mixture was stirred at room temperature for 5 d and then parti-

tioned between CH₂Cl₂and 2% aqueous citric acid. The

aqueous layer was extracted three times with CH₂Cl₂, the com-

bined organic layers were washed with brine and dried over

Na₂SO₄. Removal of the solvent and ﬂash chromatography

(SiO₂; petroleum ether/ethyl acetate, 1 : 1) afforded 25 as a col-

ourless solid, yield: 3.07 g (88%), m.p. 54–56 °C. IR (ATR): ν =

3398, 2952, 2872, 1727, 1710, 1686, 1653, 1617, 1596, 1558,

1528, 1456, 1395, 1367, 1336, 1308, 1244, 1230, 1147, 1111,

(ATR): ν = 3304, 2931, 2864, 1726, 1674, 1595, 1537, 1512,

1450, 1395, 1368, 1337, 1305, 1243, 1152, 1088, 1057, 1001,

843, 815, 740, 721, 697 cm⁻¹. ¹H NMR (500 MHz, CDCl₃): δ =

0.84 (d, J = 6.6 Hz, 3 H), 0.89 (d, J = 6.6 Hz, 3 H), 1.29 (s,

9 H), 1.37–1.47 (m, 1 H), 1.51–1.67 (m, 4 H), 1.85–1.99 (m,

2 H), 2.40 (s, 3 H), 2.94 (dd, J = 14.7, 7.4 Hz, 1 H), 3.10 (dd,

J = 14.7, 7.6 Hz, 1 H), 3.33 (q, J = 6.7 Hz, 2 H), 3.62 (s, 2 H),

4.28 (t, J = 7.4 Hz, 1 H), 4.46 (s, 2 H), 5.13 (s, 2 H), 6.66 (br t,

J = 5.6 Hz, 1 H), 6.89 (d, J = 8.7 Hz, 2 H), 7.23 (d, J = 8.7 Hz,

2 H), 7.27 (d, J = 8.5 Hz, 2 H), 7.30–7.37 (m, 5 H), 7.71 (d, J =

8.3 Hz, 2 H). ¹³C NMR and DEPT (125 MHz, CDCl₃): δ =

20.51 (CH₃), 20.57 (CH₃), 21.61 (CH₃), 23.89 (CH₂), 27.92 (3

CH₃), 28.27 (CH), 29.10 (CH₂), 30.61 (CH₂), 38.93 (CH₂),

40.49 (CH₂), 53.88 (CH₂), 60.74 (CH), 66.81 (CH₂), 67.55

(CH₂), 82.08 (C), 114.96 (2 CH), 127.65 (C), 127.74 (2 CH),

128.31 (2 CH), 128.41 (CH), 128.70 (2 CH), 129.57 (2 CH),

130.81 (2 CH), 135.90 (C), 137.34 (C), 143.32 (C), 156.51 (C),

168.25 (CvO), 169.83 (CvO), 171.67 (CvO). ESI-MS: m/z =

695.4 [M + H]⁺, 712.6 [M + NH₄]⁺.

1

1088, 1013, 951, 866, 841, 816, 762, 718, 706, 661 cm⁻¹. H

NMR (600 MHz, CDCl₃): δ = 0.84 (d, J = 6.6 Hz, 3 H), 0.90 (d,

J = 6.6 Hz, 3 H), 1.30 (s, 9 H), 1.36–1.40 (m, 1 H), 1.46–1.63

(m, 4 H), 1.85–1.99 (m, 2 H), 2.40 (s, 3 H), 2.94 (dd, J = 14.6,

7.7 Hz, 1 H), 3.10 (dd, J = 14.6, 7.7 Hz, 1 H), 3.17 (q, J =

6.7 Hz, 2 H), 4.25 (t, J = 7.3 Hz, 1 H), 4.79 (br s, 1 H), 5.09 (s,

2 H), 7.27 (d, J = 7.2 Hz, 2 H), 7.29–7.38 (m, 5 H), 7.71 (d, J =

8.3 Hz, 2 H). ¹³C NMR and DEPT (150 MHz, CDCl₃): δ =

20.52 (2 CH₃), 21.61 (CH₃), 23.82 (CH₂), 27.95 (3 CH₃), 28.37

(CH), 29.53 (CH₂), 30.62 (CH₂), 40.91 (CH₂), 53.91 (CH₂),

60.84 (CH), 66.73 (CH₂), 82.08 (C), 127.75 (2 CH), 128.22

(CH), 128.24 (CH), 128.66 (2 CH), 129.58 (3 CH), 136.79 (C),

137.44 (C), 143.30 (C), 156.51 (CvO), 169.89 (CvO).

ESI-MS: m/z = 547.8 [M + H]⁺, 564.7 [M + NH₄]⁺.

28: (S)-tert-Butyl 6-(2-(4-(2-benzyloxy-2-oxoethyl)phenoxy)-

acetamido)-2-(N-isobutyl-4-methylphenylsulfonamido)hexanoate

(0.143 g, 0.206 mmol) and palladium on activated carbon

(14 mg, 10 wt%) in ethyl acetate (10 mL) were stirred under

hydrogen (1 atm) at room temperature for 2 h. Filtration of the

reaction mixture through a short path of Celite® and removal of

the solvent afforded 28 as a colourless solid, yield: 0.125 g

(100%), m.p. 47–49 °C. IR (ATR): ν = 3364, 2957, 2933, 2870,

1725, 1641, 1611, 1569, 1555, 1512, 1449, 1394, 1367, 1337,

1291, 1239, 1152, 1089, 1062, 1000, 940, 843, 813, 720,

26: Compound 25 (0.101 g, 0.185 mmol) and palladium on

activated carbon (50 mg, 10 wt%) in ethyl acetate (15 mL) were

stirred under hydrogen (1 atm) at room temperature for 24 h. Fil-

tration of the reaction mixture through a short path of Celite®

and removal of the solvent afforded 26 as a light yellow oil,

yield: 75.4 mg (99%). IR (ATR): ν = 2987, 2932, 2870, 1727,

1673, 1654, 1598, 1534, 1493, 1464, 1451, 1394, 1367, 1338,

1

664 cm⁻¹. H NMR (500 MHz, CDCl₃/methanol-d₄, 3 : 1): δ =

0.69 (d, J = 6.6 Hz, 3 H), 0.75 (d, J = 6.6 Hz, 3 H), 1.15 (s,

9 H), 1.21–1.31 (m, 2 H), 1.37–1.51 (m, 3 H), 1.71–1.83 (m,

2 H), 2.26 (s, 3 H), 2.79 (dd, J = 14.7, 7.4 Hz, 1 H), 2.94 (dd, J

= 14.7, 7.6 Hz, 1 H), 3.15 (td, J = 7.0, 2.5 Hz, 2 H), 3.41 (s,

2 H), 4.09 (t, J = 7.4 Hz, 1 H), 4.30 (s, 2 H), 6.76 (d, J =

8.7 Hz, 2 H), 7.08 (d, J = 8.7 Hz, 2 H), 7.15 (d, J = 8.2 Hz,

2 H), 7.55 (d, J = 8.2 Hz, 2 H). ¹³C NMR and DEPT (125 MHz,

CDCl₃/methanol-d₄, 3 : 1): δ = 19.90 (CH₃), 19.97 (CH₃), 21.00

(CH₃), 23.45 (CH₂), 27.34 (3 CH₃), 27.81 (CH), 28.46 (CH₂),

30.10 (CH₂), 38.49 (CH₂), 39.95 (CH₂), 53.55 (CH₂), 60.43

(CH), 66.89 (CH₂), 81.95 (C), 114.47 (2 CH), 127.23 (2 CH),

127.75 (C), 129.27 (2 CH), 130.33 (2 CH), 136.60 (C), 143.28

(C), 156.03 (C), 168.74 (CvO), 169.50 (CvO), 174.08

(CvO). ESI-MS: m/z = 603.1 [M − H]⁻.

1

1250, 1151, 1089, 996, 937, 845, 814, 719, 664, 656 cm⁻¹. H

NMR (500 MHz, CDCl₃/methanol-d₄, 3 : 1): δ = 0.66 (d, J = 6.6

Hz, 3 H), 0.71 (d, J = 6.7 Hz, 3 H), 1.11 (s, 9 H), 1.14–1.20 (m,

2 H), 1.22–1.34 (m, 2 H), 1.36–1.46 (m, 1 H), 1.65–1.80 (m,

2 H), 2.22 (s, 3 H), 2.44 (t, J = 7.2 Hz, 2 H), 2.75 (dd, J = 14.7,

7.4 Hz, 1 H), 2.90 (dd, J = 14.7, 7.6 Hz, 1 H), 4.04 (t, J =

7.4 Hz, 1 H), 7.11 (d, J = 8.2 Hz, 2 H), 7.51 (d, J = 8.2 Hz,

2 H). ¹³C NMR and DEPT (125 MHz, CDCl₃/methanol-d₄,

3 : 1): δ = 20.48 (CH₃), 20.53 (CH₃), 21.54 (CH₃), 24.14 (CH₂),

27.92 (3 CH₃), 28.43 (CH), 30.91 (CH₂), 32.71 (CH₂), 41.56

(CH₂), 54.17 (CH₂), 61.21 (CH), 82.55 (C), 127.84 (2 CH),

129.88 (2 CH), 137.25 (C), 143.93 (C), 170.18 (CvO).

ESI-MS: m/z = 413.4 [M + H]⁺.

Benzyl 4-(2-tert-butoxy-2-oxoethoxy)phenylacetate. tert-Butyl

bromoacetate (4.00 mL, 27.1 mmol) was added dropwise to a

mixture of benzyl 4-hydroxyphenylacetate (6.57 g, 27.1 mmol)

and Cs₂CO₃(13.3 g, 40.7 mmol) in DMF (60 mL). The reaction

mixture was stirred at room temperature for 5 h. The reaction

mixture was diluted with ethyl acetate and washed four times

with water and once with brine. The organic layer was dried over

Na₂SO₄. Removal of the solvent and ﬂash chromatography

(SiO₂; pentane/ethyl acetate, 20 : 1 to 3 : 2) afforded benzyl 4-(2-

tert-butoxy-2-oxoethoxy)phenylacetate as a colourless oil, yield:

9.15 g (95%). IR (ATR): ν = 3065, 3031, 2978, 2934, 2056,

1918, 1869, 1844, 1828, 1727, 1651, 1611, 1558, 1541, 1512,

(S)-tert-Butyl 6-(2-(4-(2-benzyloxy-2-oxoethyl)phenoxy)acet-

amido)-2-(N-isobutyl-4-methylphenylsulfonamido)hexanoate. A

catalytic amount of DMAP was added to a solution of 26

(92.3 mg, 0.224 mmol), 27 (0.100 g, 0.333 mmol) and

EDC·HCl (64.3 mg, 0.335 mmol) in CH₂Cl₂(10 mL). The reac-

tion mixture was stirred at room temperature for 5 d, then diluted

with CH₂Cl₂, washed three times with H₂O, once with brine and

dried over Na₂SO₄. Removal of the solvent and ﬂash chromato-

graphy (SiO₂; pentane/ethyl acetate, 5 : 1 to 2 : 3) afforded

(S)-tert-butyl

6-(2-(4-(2-benzyloxy-2-oxoethyl)phenoxy)acet-

amido)-2-(N-isobutyl-4-methylphenylsulfonamido)hexanoate as

a colourless solid, yield: 87.4 mg (56%), m.p. 72–74 °C. IR

1454, 1369, 1303, 1216, 1145, 1079, 982, 842, 737, 697 cm⁻¹

.

¹H NMR (500 MHz, CDCl₃): δ = 1.49 (s, 9 H), 3.61 (s, 2 H),

Org. Biomol. Chem., 2012, 10, 8216–8235 | 8229

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4.50 (s, 2 H), 5.12 (s, 2 H), 6.85 (d, J = 8.7 Hz, 2 H), 7.20 (d,

J = 8.7 Hz, 2 H), 7.30–7.37 (m, 5 H). ¹³C NMR and DEPT

(125 MHz, CDCl₃): δ = 28.19 (3 CH₃), 40.59 (CH₂), 65.89

(CH₂), 66.74 (CH₂), 82.52 (C), 114.85 (2 CH), 127.01 (C),

128.28 (2 CH), 128.36 (CH), 128.69 (2 CH), 130.53 (2 CH),

135.98 (C), 157.21 (C), 168.16 (CvO), 171.76 (CvO).

ESI-MS: m/z = 374.2 [M + NH₄]⁺, 730.5 [2 M + NH₄]⁺.

(br s, 1 H), 13.67 (s, 1 H). ¹³C NMR and DEPT (125 MHz,

acetone-d₆): δ = 12.79 (CH₃), 40.47 (CH₂), 113.81 (2 CH),

115.85 (C), 122.67 (C), 123.50 (C), 126.15 (CH), 128.31 (C),

129.47 (CH), 131.31 (2 CH), 136.20 (C), 153.30 (C), 172.93

(CvO), one carbon signal missing. ESI-MS: m/z = 353.2

[M + H]⁺.

27: Triﬂuoroacetic acid (60 mL) was added slowly at 0 °C to a

solution of benzyl 4-(2-tert-butoxy-2-oxoethoxy)phenylacetate

(1.28 g, 3.57 mmol) in CH₂Cl₂(60 mL). The mixture was stirred

at room temperature for 90 min, diluted with CH₂Cl₂and

washed four times with water and once with brine. Drying over

Na₂SO₄and removal of the solvent afforded 27 as a light yellow

solid, yield: 0.815 g (76%), m.p. 103–104 °C. IR (ATR): ν =

3058, 3035, 2922, 2890, 2774, 1917, 1870, 1844, 1726, 1651,

1610, 1588, 1511, 1421, 1379, 1347, 1308, 1253, 1224, 1158,

tert-Butyl 4-hydroxyphenylacetate (35). A mixture of 4-hydro-

xyphenylacetic acid (34) (2.00 g, 13.2 mmol), tert-butyl aceto-

acetate (14 mL, 84.4 mmol) and conc. H₂SO₄(0.104 g,

1.06 mmol) was stirred in a sealed tube at room temperature for

45 h and then diluted with diethyl ether. The organic layer was

washed twice with H₂O, twice with NaHCO₃(dil. aqueous),

once with brine and dried over MgSO₄. Removal of the solvent

and ﬂash chromatography (SiO₂; pentane/ethyl acetate, 8 : 1 to

6 : 1) afforded tert-butyl 4-hydroxyphenylacetate (35) as a col-

ourless solid, yield: 1.14 g (41%), m.p. 92–93 °C. IR (ATR): ν =

3306, 3007, 2980, 2934, 2056, 2030, 2009, 1917, 1871, 1844,

1771, 1734, 1717, 1693, 1653, 1613, 1593, 1559, 1515, 1446,

1369, 1313, 1269, 1221, 1166, 1129, 954, 859, 826, 808, 767,

1

1083, 984, 940, 907, 838, 807, 774, 741, 694 cm⁻¹. H NMR

(600 MHz, CDCl₃): δ = 3.62 (s, 2 H), 4.67 (s, 2 H), 5.13 (s, 2

H), 6.88 (d, J = 8.6 Hz, 2 H), 7.23 (d, J = 8.6 Hz, 2 H),

7.30–7.37 (m, 5 H). ¹³C NMR and DEPT (150 MHz, CDCl₃):

δ = 40.54 (CH₂), 65.04 (CH₂), 66.84 (CH₂), 114.94 (2 CH),

127.73 (C), 128.31 (2 CH), 128.41 (CH), 128.71 (2 CH), 130.76

(2 CH), 135.91 (C), 156.67 (C), 171.74 (CvO), 173.12 (CvO).

ESI-MS: m/z = 318.1 [M + NH₄]⁺, 618.0 [2 M + NH₄]⁺, 623.1

[2 M + Na]⁺. Elemental analysis calcd for C₁₇H₁₆O₅: C 67.99, H

5.37; found: C 68.13, H 5.34.

32: A solution of 3,5-dichloro-2-hydroxyacetophenone (29)

(1.00 g, 4.88 mmol) and 4-hydrazinobenzoic acid (30) (0.742 g,

4.88 mmol) in ethanol (90 mL) and AcOH (0.25 mL) was stirred

at reﬂux for 2.5 h. During the reaction, brown crystals precipi-

tated and were collected by ﬁltration of the hot reaction mixture.

The ﬁltrate was concentrated, ethanol (30 mL) was added, the

mixture was heated at reﬂux and ﬁltered immediately. These

steps were repeated (10 mL ethanol). After drying 32 was

1

711, 665 cm⁻¹. H NMR (500 MHz, CDCl₃): δ = 1.44 (s, 9 H),

3.45 (s, 2 H), 5.15 (s, 1 H), 6.74 (d, J = 8.5 Hz, 2 H), 7.11 (d,

J = 8.5 Hz, 2 H). ¹³C NMR and DEPT (125 MHz, CDCl₃):

δ = 28.17 (3 CH₃), 41.86 (CH₂), 81.13 (C), 115.54 (2 CH),

126.67 (C), 130.50 (2 CH), 154.81 (C), 171.97 (CvO).

MS (EI): m/z (%) = 208 (13) [M⁺], 107 (93), 77 (16), 57 (100),

41 (19).

36: A mixture of tert-butyl 4-hydroxyphenylacetate (35)

(1.12 g, 5.38 mmol) and 5-ﬂuoro-2-nitrobenzaldehyde (0.915 g,

5.38 mmol), K₂CO₃(0.901 g, 6.45 mmol) in DMF (46 mL) was

stirred at 120 °C for 45 min. The reaction mixture was parti-

tioned between 2% aqueous citric acid and ethyl acetate. The

organic layer was washed three times with H₂O, once with brine

and dried over MgSO₄. Removal of the solvent and ﬂash chrom-

atography (SiO₂; pentane/ethyl acetate, 30 : 1 to 6 : 1) afforded

36 as a yellow solid, yield: 1.20 g (63%), m.p. 69–70 °C. IR

(ATR): ν = 3104, 3072, 2978, 2929, 2056, 2031, 1771, 1727,

1697, 1653, 1624, 1581, 1541, 1510, 1469, 1417, 1365, 1326,

1296, 1270, 1230, 1203, 1140, 1067, 1019, 966, 936, 878, 849,

808, 757, 681, 643 cm⁻¹. ¹H NMR (500 MHz, CDCl₃): δ = 1.46

(s, 9 H), 3.57 (s, 2 H), 7.04 (d, J = 8.5 Hz, 2 H), 7.20 (d, J =

9.0 Hz, 1 H), 7.34–7.36 (m, 3 H), 8.15 (d, J = 9.0 Hz, 1 H),

10.43 (s, 1 H). ¹³C NMR and DEPT (125 MHz, CDCl₃): δ =

28.19 (3 CH₃), 41.95 (CH₂), 81.38 (C), 116.91 (CH), 120.70

(2 CH), 127.49 (CH), 131.53 (2 CH), 131.67 (CH), 132.66 (C),

134.56 (C), 143.35 (C), 153.13 (C), 163.09 (C), 170.75 (C),

188.20 (CHO). ESI-MS: m/z = 375.2 [M + NH₄]⁺, 732.3 [2 M +

NH₄]⁺. Elemental analysis calcd for C₁₉H₁₉NO₆: C 63.86,

H 5.36, N 3.92; found: C 63.92, H 5.37, N 3.91.

38: NaBH₃CN (0.137 g, 2.18 mmol) was added to a solution

of 36 (0.688 g, 1.93 mmol) and 37 (0.540 g, 1.93 mmol) in

AcOH (114 μL, 1.99 mmol) and CH₂Cl₂(20 mL). The reaction

mixture was stirred at room temperature for 17 h and then treated

with 1 M HCl (12 mL) for 10 min. After addition of 1 M NaOH

(16 mL), the layers were separated, and the aqueous layer was

extracted twice with CH₂Cl₂. The combined organic layers were

washed with brine and dried over MgSO₄. Removal of the

solvent and ﬂash chromatography (SiO₂; pentane/ethyl acetate,

10 : 1 to 2 : 3) afforded 38 as a yellow, viscous oil, yield: 0.998 g

obtained as brown crystals, yield: 1.09

g (66%), m.p.

299–301 °C. IR (ATR): ν = 3379, 2078, 2790, 2530, 2053,

1661, 1600, 1577, 1518, 1451, 1420, 1290, 1251, 1163, 1120,

939, 839, 766, 737, 684 cm⁻¹. ¹H NMR (500 MHz, DMSO-d₆):

δ = 2.43 (s, 3 H), 7.12 (d, J = 8.7 Hz, 2 H), 7.55 (s, 1 H), 7.60

(s, 1 H), 7.91 (d, J = 8.7 Hz, 2 H), 10.21 (s, 1 H), 12.50 (br s,

1 H), 13.56 (s, 1 H). ¹³C NMR and DEPT (125 MHz, DMSO-d₆):

δ = 13.63 (CH₃), 111.88 (2 CH), 121.33 (C), 122.03 (C), 122.47

(C), 125.99 (CH), 129.03 (CH), 131.38 (2 CH), 147.62 (C),

148.00 (C), 152.02 (C), 167.06 (CvO, CvN). ESI-MS: m/z =

339.1 [M + H]⁺. Elemental analysis calcd for C₁₅H₁₂Cl₂N₂O₃:

C 53.12, H 3.57, N 8.26; found: C 52.86, H 3.54, N 8.18.

33: A solution of 3,5-dichloro-2-hydroxyacetophenone (29)

(1.00 g, 4.88 mmol) and 4-hydrazinophenylacetic acid³⁶(31)

(0.811 g, 4.87 mmol) in ethanol (90 mL) and AcOH (0.25 mL)

was stirred and heated at reﬂux for 2.5 h. The solvent was

removed and the residue recrystallised from ethyl acetate

(100 mL) and i-hexane (600 mL). After drying 33 was obtained

as orange crystals, yield: 0.881 g (51%), m.p. 231–233 °C. IR

(ATR): ν = 3371, 3077, 3031, 2906, 2731, 2635, 2566, 2056,

1733, 1704, 1652, 1619, 1585, 1517, 1454, 1424, 1366, 1301,

1244, 1198, 1174, 1116, 954, 853, 822, 799, 736 cm⁻^{1 1}H

.

NMR (500 MHz, acetone-d₆): δ = 2.46 (s, 3 H), 3.58 (s, 2 H),

7.09 (d, J = 8.5 Hz, 2 H), 7.30 (d, J = 8.5 Hz, 2 H), 7.38 (d, J =

2.4 Hz, 1 H), 7.52 (d, J = 2.4 Hz, 1 H), 9.16 (s, 1 H), 10.71

8230 | Org. Biomol. Chem., 2012, 10, 8216–8235

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(83%). IR (ATR): ν = 3432, 2924, 2852, 2056, 2030, 1843,

1729, 1611, 1581, 1558, 1540, 1515, 1473, 1448, 1419, 1365,

1339, 1256, 1226, 1141, 1101, 1017, 957, 892, 830, 754,

between 2% aqueous citric acid and CH₂Cl₂, washed with 2%

citric acid, H₂O and brine. The organic layer was dried over

Na₂SO₄and evaporated. The residue was puriﬁed by ﬂash

chromatography (SiO₂; pentane/[CH₂Cl₂/methanol 10 : 1], 10 : 1

to 2 : 3) to yield a colourless solid, yield: 0.249 g (61%). This

solid (0.139 g, 0.185 mmol) was dissolved in CH₂Cl₂(5 mL).

CF₃COOH (5 mL) was added at 0 °C, and the mixture stirred at

room temperature for 90 min, diluted with CH₂Cl₂and washed

four times with water and once with brine. After drying over

Na₂SO₄and removal of the solvent, compound 44 was obtained

as a colourless solid, yield: 0.118 g (93%), m.p. 152–154 °C. IR

(ATR): ν = 3272, 3067, 2931, 2857, 1770, 1694, 1632, 1583,

1533, 1490, 1475, 1447, 1387, 1340, 1226, 1156, 1050, 951,

694 cm⁻^{1 1}H NMR (500 MHz, CDCl₃): δ = 0.98–1.85 (m,

.

32 H), 2.24–2.36 (m, 2 H), 2.42–2.53 (m, 1 H), 2.78 (s, 0.4 ×

3 H), 2.80 (s, 0.6 × 3 H), 3.54 (s, 2 H), 3.57–3.60 (m, 0.6 H),

3.96–4.05 (m, 2 H), 4.40–4.44 (m, 0.4 H), 6.80–6.84 (m, 1 H),

7.02 (d, J = 8.5 Hz, 2 H), 7.23 (d, J = 7.6 Hz, 1 H), 7.30 (d, J =

8.5 Hz, 2 H), 7.94 (d, J = 9.0 Hz, 1 H). ESI-MS: m/z = 622.6

[M + H]⁺, 1243.8 [2 M + H]⁺.

39: Compound 38 (0.859 g, 1.38 mmol) and palladium on

activated carbon (0.127 g, 10 wt%) in methanol (14 mL) were

stirred under hydrogen (1 atm) at room temperature for 2 h. Fil-

tration of the reaction mixture through a short path of Celite®

and removal of the solvent afforded 39 as a yellow oil, yield:

0.772 g (94%). IR (ATR): ν = 3415, 3321, 2924, 2852, 2056,

2026, 2007, 1727, 1684, 1624, 1576, 1558, 1541, 1447, 1366,

1

891, 823, 778, 757, 739, 676, 648 cm⁻¹. H NMR (500 MHz,

methanol-d₄): δ = 1.11–1.20 (m, 1 H), 1.26–1.40 (m, 4 H),

1.63–2.05 (m, 8 H), 2.11–2.16 (m, 2 H), 2.19–2.28 (m, 1 H),

2.31–2.44 (m, 2 H), 2.47–2.55 (m, 1 H), 2.85–2.91 (m, 1 H),

3.65–3.76 (m, 2 H), 4.15–4.40 (m, 5 H), 7.15–7.23 (m, 3 H),

7.28–7.32 (m, 2 H), 7.37–7.41 (m, 2 H), 7.47 (q, J = 6.6 Hz,

1 H), 7.63–7.69 (m, 2 H), 7.79–7.83 (m, 2 H). ¹³C NMR and

DEPT (125 MHz, methanol-d₄): δ = 26.07 (2 CH₂), 26.42

(CH₂), 28.11 (CH₂), 28.34 (CH₂), 29.68 (CH₂), 29.89 (CH₂),

33.13 (CH₂), 34.92 (CH₂), 38.88 (CH and CH₂), 42.57 (CH₂),

54.07 (CH), 54.77 (CH), 66.27 (C), 68.22 (CH₂), 117.22 (d, J =

21.2 Hz, CH), 119.65 (d, J = 22.0 Hz, CH), 121.00 (CH),

121.05 (CH), 126.17 (CH), 126.38 (CH), 128.18 (CH), 128.24

(CH), 128.46 (CH), 128.88 (2 CH), 132.01 (CH), 140.54 (C),

142.57 (C), 142.62 (C), 144.97 (C), 145.44 (C), 158.80 (CvO),

164.40 (d, J = 247.3 Hz, C), 168.09 (CvN), 172.49 (2 CvO),

175.41 (CvO). ESI-MS: m/z = 696.5 [M + H]⁺.

1

1295, 1222, 1138, 1101, 1015, 954, 888, 810, 755 cm⁻¹. H

NMR (500 MHz, CDCl₃): δ = 0.99–1.85 (m, 34 H), 2.24–2.37

(m, 2 H), 2.38–2.55 (m, 1 H), 2.78 (s, 0.4 × 3 H), 2.79 (s, 0.6 ×

3 H), 3.47 (s, 2 H), 3.48–3.51 (m, 0.4 H), 3.68–3.76 (m, 2 H),

4.40–4.45 (m, 0.6 H), 6.62 (dd, J = 8.2, 3.4 Hz, 1 H), 6.76–6.81

(m, 2 H), 6.85 (d, J = 8.6 Hz, 2 H), 7.16 (d, J = 8.6 Hz, 2 H).

ESI-MS: m/z = 592.5 [M + H]⁺.

40: A solution of 39 (0.888 g, 1.50 mmol) and cyanogen

bromide (0.240 g, 2.27 mmol) in ethanol (30 mL) was stirred at

reﬂux for 17 h. After removal of the solvent, the residue was

puriﬁed by ﬂash chromatography (SiO₂; pentane/[CH₂Cl₂/

methanol 10 : 1], 5 : 1 to 2 : 3) to afford 40 as a light brown

solid, yield: 0.775 g (84%), m.p. 112–113 °C. IR (ATR): ν =

3127, 2972, 2926, 2853, 1844, 1726, 1658, 1618, 1557, 1496,

1448, 1414, 1366, 1314, 1236, 1216, 1140, 1102, 1015, 955,

875, 825 cm⁻¹. ¹H NMR (500 MHz, CDCl₃): δ = 0.85–1.87 (m,

32 H), 2.02–2.18 (m, 2 H), 2.59–2.71 (m, 1 H), 2.80 (s, 0.4 ×

3 H), 2.86 (s, 0.6 × 3 H), 3.51 (s, 2 H), 4.11–4.21 (m, 2.4 H),

4.34–4.45 (m, 0.6 H), 6.70 (d, J = 3.1 Hz, 1 H), 6.02–6.94 (m,

3 H), 7.19 (d, J = 8.7 Hz, 1 H), 7.22 (d, J = 8.5 Hz, 2 H), 8.17

(br s, 2 H). ESI-MS: m/z = 617.6 [M + H]⁺.

45: EtN(i-Pr)₂(375 μL, 2.21 mmol) was added to a solution

of Fmoc-Glu-OtBu (43) (0.910 g, 2.14 mmol) and HATU

(0.814 g, 2.14 mmol) in CH₂Cl₂(50 mL). The mixture was

stirred at room temperature for 10 min and then 4-(tert-butyl-

dimethylsilyloxymethyl)aniline (0.495 g, 2.08 mmol) and EtN-

(i-Pr)₂(375 μL, 2.21 mmol) were added. After stirring at room

temperature for 28 h, the mixture was partitioned between 2%

aqueous citric acid and CH₂Cl₂, washed with 2% citric acid,

H₂O and brine. The organic layer was dried over Na₂SO₄.

Removal of the solvent and ﬂash chromatography (SiO₂;

pentane/ethyl acetate, 10 : 1 to 3 : 7) afforded the amide as a col-

ourless solid, yield: 1.07 g (80%), m.p. 99–100 °C. IR (ATR):

ν = 3326, 2953, 2930, 2888, 2856, 1771, 1733, 1694, 1662,

1601, 1557, 1528, 1452, 1410, 1369, 1338, 1250, 1155, 1081,

41: A mixture of 40 (0.245 g, 0.397 mmol), silica gel (2.0 g)

and toluene (13 mL) was stirred at reﬂux for 3.5 h. The reaction

mixture was diluted with CH₂Cl₂/methanol (10 : 1) and ﬁltered

through a short path of Celite®. After removal of the solvent,

compound 41 was obtained as a light yellow solid, yield:

0.220 g (99%), m.p. 145–146 °C. IR (ATR): ν = 3306, 3121,

2924, 2853, 2029, 1724, 1658, 1616, 1558, 1495, 1447, 1410,

1056, 938, 835, 775, 758, 737, 664, 621 cm⁻^{1 1}H NMR

.

1314, 1235, 1214, 1166, 1146, 1099, 1017, 955, 891, 803 cm⁻¹

.

(500 MHz, CDCl₃): δ = 0.08 (s, 6 H), 0.93 (s, 9 H), 1.46 (s,

9 H), 1.87–1.95 (m, 1 H), 2.27–2.41 (m, 3 H), 4.20 (t, J =

6.8 Hz, 1 H), 4.26–4.30 (m, 1 H), 4.39–4.50 (m, 2 H), 4.68 (s,

2 H), 5.61 (br d, J = 8.2 Hz, 1 H), 7.25–7.33 (m, 4 H),

7.38–7.42 (m, 2 H), 7.55 (d, J = 8.3 Hz, 2 H), 7.60 (t, J =

6.5 Hz, 2 H), 7.77 (dd, J = 7.5, 2.4 Hz, 2 H), 8.29 (br s, 1 H).

¹³C NMR and DEPT (125 MHz, CDCl₃): δ = −5.09 (2 CH₃),

18.54 (C), 26.09 (3 CH₃), 28.11 (3 CH₃), 30.33 (CH₂), 34.10

(CH₂), 47.33 (CH), 53.85 (CH), 64.78 (CH₂), 67.23 (CH₂),

83.08 (C), 119.67 (2 CH), 120.14 (CH), 120.18 (CH), 125.15

(CH), 125.26 (CH), 126.87 (2 CH), 127.23 (2 CH), 127.91

(2 CH), 137.11 (C), 137.36 (C), 141.46 (C), 141.49 (C), 143.69

(C), 143.92 (C), 157.03 (CvO), 170.39 (CvO), 171.14

(CvO). ESI-MS: m/z = 645.5 [M + H]⁺, 662.5 [M + NH₄]⁺.

¹H NMR (500 MHz, CDCl₃): δ = 0.86–1.87 (m, 23 H),

2.01–2.06 (m, 0.5 × 2 H), 2.18–2.22 (m, 0.5 × 2 H), 2.59–2.65

(m, 1 H), 2.79 (s, 0.5 × 3 H), 2.85 (s, 0.5 × 3 H), 3.47–3.53 (m,

0.5 H), 3.61 (s, 2 H), 4.10–4.24 (m, 2 H), 4.32–4.35 (m, 0.5 H),

6.70–6.74 (m, 1 H), 6.86–6.93 (m, 3 H), 7.13 (d, J = 8.4 Hz,

1 H), 7.22–7.25 (m, 2 H), 8.11 (br s, 2 H), 11.58 (br s, 1 H).

ESI-MS: m/z = 561.5 [M + H]⁺.

44: To a solution of Fmoc-Glu-OtBu (43) (0.430 g,

1.01 mmol) and HATU (0.384 g, 1.01 mmol) in CH₂Cl₂

(15 mL) was added EtN(i-Pr)₂(172 μL, 1.01 mmol). After stir-

ring at room temperature for 10 min, 42 (0.224 g, 0.537 mmol)

and EtN(i-Pr)₂(223 μL, 1.31 mmol) were added. After stirring

for 18 h at room temperature, the mixture was partitioned

Org. Biomol. Chem., 2012, 10, 8216–8235 | 8231

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HF–pyridine (5.0 mL) was added at 0 °C to a solution of this

silyl ether (0.203 g, 0.315 mmol) in THF (10 mL). The reaction

mixture was stirred at 0 °C for 75 min, diluted with ethyl acetate

and neutralised with Na₂CO₃(sat. aqueous). The organic layer

was washed with 2% aqueous citric acid, H₂O, brine and dried

over Na₂SO₄. After removal of the solvent, the residue was

puriﬁed by ﬂash chromatography (SiO₂; petroleum ether/

[CH₂Cl₂/methanol 10 : 1], 10 : 1 to 1 : 1), affording the alcohol

as a colourless solid, yield: 0.145 g (87%), m.p. 140–142 °C. IR

(ATR): ν = 3311, 3253, 3121, 3058, 2931, 2856, 1695, 1662,

1595, 1534, 1447, 1414, 1367, 1337, 1257, 1227, 1153, 1081,

1458, 1411, 1367, 1309, 1255, 1145, 1083, 1007, 939, 834, 775,

1

665 cm⁻¹. H NMR (500 MHz, CDCl₃): δ = 0.08 (s, 6 H), 0.93

(s, 9 H), 1.46 (s, 9 H), 2.01 (quin, J = 7.2 Hz, 2 H), 2.34 (t, J =

7.0 Hz, 2 H), 2.41 (t, J = 7.3 Hz, 2 H), 4.69 (s, 2 H), 7.27 (d,

J = 8.4 Hz, 2 H), 7.48 (d, J = 8.4 Hz, 2 H). ¹³C NMR and DEPT

(125 MHz, CDCl₃): δ = −5.08 (2 CH₃), 18.55 (C), 21.25 (CH₂),

26.09 (3 CH₃), 28.26 (3 CH₃), 34.55 (CH₂), 36.72 (CH₂), 64.78

(CH₂), 80.80 (C), 119.74 (2 CH), 126.91 (2 CH), 136.85 (C),

137.46 (C), 170.72 (CvO), 173.01 (CvO). ESI-MS: m/z =

408.3 [M + H]⁺, 815.7 [2 M + H]⁺.

TBAF (1 ^Min THF, 180 μL, 1.80 mmol) was added at 0 °C to

a solution of this amide (0.503 g, 1.23 mmol) in THF (25 mL).

The reaction mixture was stirred at room temperature for 1 h and

then diluted with ethyl acetate. The organic layer was washed

with H₂O, brine and dried over Na₂SO₄. After removal of the

solvent, the residue was puriﬁed by ﬂash chromatography (SiO₂;

petroleum ether/ethyl acetate, 4 : 1 to 1 : 1), affording the alcohol

as a colourless, viscous oil, yield: 0.348 g (96%). ¹H NMR

(500 MHz, CDCl₃): δ = 1.45 (s, 9 H), 1.97 (quin, J = 7.2 Hz,

2 H), 2.15 (br s, 1 H), 2.31 (t, J = 7.1 Hz, 2 H), 2.38 (t, J =

7.4 Hz, 2 H), 4.62 (s, 2 H), 7.27 (d, J = 8.4 Hz, 2 H), 7.47 (d,

J = 8.4 Hz, 2 H), 7.81 (br s, 1 H). ¹³C NMR and DEPT

(125 MHz, CDCl₃): δ = 21.20 (CH₂), 28.23 (3 CH₃), 34.56

(CH₂), 36.62 (CH₂), 64.97 (CH₂), 80.89 (C), 120.11 (2 CH),

127.91 (2 CH), 136.88 (C), 137.49 (C), 171.03 (CvO), 173.11

(CvO). ESI-MS: m/z = 294.2 [M + H]⁺.

1

1054, 1033, 960, 892, 827, 784, 755, 733, 666 cm⁻¹. H NMR

(500 MHz, CDCl₃): δ = 1.46 (s, 9 H), 1.87–1.95 (m, 1 H),

2.27–2.41 (m, 3 H), 4.18 (t, J = 6.8 Hz, 1 H), 4.25–4.30 (m,

1 H), 4.38–4.49 (m, 2 H), 4.62 (s, 2 H), 5.60 (br d, J = 8.0 Hz,

1 H), 7.29–7.33 (m, 4 H), 7.38–7.42 (m, 2 H), 7.57–7.61 (m,

4 H), 7.77 (dd, J = 7.5, 2.9 Hz, 2 H), 8.35 (br s, 1 H). ¹³C NMR

and DEPT (125 MHz, CDCl₃): δ = 28.12 (3 CH₃), 30.39 (CH₂),

34.16 (CH₂), 47.33 (CH), 53.86 (CH), 65.15 (CH₂), 67.26

(CH₂), 83.15 (C), 119.92 (2 CH), 120.16 (CH), 120.20 (CH),

125.15 (CH), 125.36 (CH), 127.24 (2 CH), 127.94 (4 CH),

136.74 (C), 137.84 (C), 141.46 (C), 141.48 (C), 143.69 (C),

143.90 (C), 157.07 (CvO), 170.53 (CvO), 171.09 (CvO).

ESI-MS: m/z = 531.5 [M + H]⁺, 553.5 [M + Na]⁺.

A mixture of this alcohol (0.599 g, 1.13 mmol) and manga-

nese dioxide (0.981 g, 11.3 mmol) in CH₂Cl₂(60 mL) was

stirred at room temperature for 22 h. The reaction mixture was

ﬁltered through a short path of Celite®. After removal of the

solvent, the residue was puriﬁed by ﬂash chromatography (SiO₂;

petroleum ether/[CH₂Cl₂/methanol 10 : 1], 10 : 1 to 3 : 1), afford-

ing 45 as a colourless solid, yield: 0.484 g (81%), m.p.

104–105 °C. IR (ATR): ν = 3302, 3061, 2978, 2936, 1718,

1692, 1670, 1590, 1533, 1447, 1414, 1368, 1306, 1260, 1216,

A mixture of this alcohol (0.348 g, 1.19 mmol) and manga-

nese dioxide (1.02 g, 11.7 mmol) in CH₂Cl₂(60 mL) was stirred

at room temperature for 22 h. The reaction mixture was ﬁltered

through a short path of Celite®. After removal of the solvent, the

residue was puriﬁed by ﬂash chromatography (SiO₂; petroleum

ether/ethyl acetate, 4 : 1 to 2 : 1), affording 46 as a colourless

solid, yield: 0.257 g (74%), m.p. 131–132 °C. IR (ATR): ν =

3438, 3326, 3064, 3035, 2952, 2896, 2056, 2030, 1771, 1727,

1699, 1673, 1653, 1581, 1493, 1449, 1401, 1387, 1340, 1223,

1206, 1172, 1017, 1001, 972, 898, 853, 807, 771, 702,

1

1151, 1102, 1086, 1057, 1031, 963, 829, 734, 642 cm⁻¹. H

NMR (300 MHz, CDCl₃): δ = 1.46 (s, 9 H), 1.82–1.95 (m, 1 H),

2.28–2.43 (m, 3 H), 4.19–4.29 (m, 2 H), 4.42–4.55 (m, 2 H),

5.63 (br d, J = 8.0 Hz, 1 H), 7.32 (t, J = 7.0 Hz, 2 H), 7.41 (td,

J = 7.4, 2.8 Hz, 2 H), 7.59 (d, J = 7.4 Hz, 2 H), 7.76–7.85 (m,

6 H), 8.98 (br s, 1 H), 9.90 (s, 1 H). ¹³C NMR and DEPT

(75 MHz, CDCl₃): δ = 28.11 (3 CH₃), 30.81 (CH₂), 34.34

(CH₂), 47.34 (CH), 53.68 (CH), 67.41 (CH₂), 83.43 (C), 119.39

(2 CH), 120.24 (2 CH), 125.08 (CH), 125.16 (CH), 127.23

(CH), 127.27 (CH), 128.01 (2 CH), 131.26 (2 CH), 132.28 (C),

141.49 (C), 141.54 (C), 143.54 (C), 143.83 (C), 143.99 (C),

157.39 (CO), 170.86 (CvO), 171.04 (CvO), 191.17 (CHO).

ESI-MS: m/z = 529.4 [M + H]⁺, 546.4 [M + NH₄]⁺.

1

682 cm⁻¹. H NMR (500 MHz, CDCl₃): δ = 1.46 (s, 9 H), 2.02

(quin, J = 6.7 Hz, 2 H), 2.36 (t, J = 6.9 Hz, 2 H), 2.47 (t, J =

7.3 Hz, 2 H), 7.72 (d, J = 8.6 Hz, 2 H), 7.84 (d, J = 8.6 Hz,

2 H), 8.06 (br s, 1 H), 9.91 (s, 1 H). ¹³C NMR and DEPT

(125 MHz, CDCl₃): δ = 21.04 (CH₂), 28.24 (3 CH₃), 34.43

(CH₂), 36.82 (CH₂), 81.10 (C), 119.29 (2 CH), 131.32 (2 CH),

132.27 (C), 143.78 (C), 171.34 (CvO), 173.16 (CvO), 191.23

(CHO). ESI-MS: m/z = 292.1 [M + H]⁺. Elemental analysis

calcd for C₁₆H₂₁NO₂: C 65.96, H 7.27, N 4.81; found: C 66.19,

H 7.40, N 5.04.

46: EtN(i-Pr)₂(468 μL, 2.75 mmol) was added to a solution

of mono-tert-butyl glutarate (0.622 g, 3.30 mmol) and HATU

(1.26 g, 3.31 mmol) in CH₂Cl₂(25 mL). The mixture was stirred

at room temperature for 10 min and then 4-(tert-butyldimethyl-

silyloxymethyl)aniline (0.523 g, 2.21 mmol) and EtN(i-Pr)₂

(468 μL, 2.75 mmol) were added. After stirring at room tempera-

ture for 24 h, the mixture was partitioned between 2% aqueous

citric acid and CH₂Cl₂. The organic layer was washed with 2%

citric acid, H₂O and brine. The organic layer was dried over

Na₂SO₄. Removal of the solvent and ﬂash chromatography

(SiO₂; pentane/ethyl acetate, 20 : 1 to 3 : 7) afforded the amide as

a colourless, viscous oil, yield: 0.702 g (78%). IR (ATR): ν =

3296, 2954, 2930, 2856, 1727, 1699, 1661, 1602, 1534, 1472,

47: NaBH(OAc)₃(0.306 g, 1.44 mmol) and a solution of 45

(0.381 g, 0.721 mmol) in 1,2-C₂H₄Cl₂(8 mL) were added to a

solution of 42 (0.200 g, 0.480 mmol) and EtN(i-Pr)₂(122 μL,

0.717 mmol) in 1,2-C₂H₄Cl₂(8 mL). The reaction mixture was

stirred for 18 h at room temperature and then partitioned

between Na₂CO₃(sat. aqueous) and CH₂Cl₂. The organic layer

was washed with H₂O, brine and dried over Na₂SO₄. Removal

of the solvent and puriﬁcation of the residue by ﬂash chromato-

graphy (SiO₂; pentane/[CH₂Cl₂/methanol 10 : 1], 10 : 1 to 2 : 3)

afforded 47 as a colourless solid, yield: 0.289 g (70%), m.p.

114–116 °C. IR (ATR): ν = 3278, 3061, 2930, 2855, 1696,

1687, 1531, 1449, 1368, 1339, 1307, 1243, 1152, 1078, 1046,

893, 844, 824, 781, 738, 696 cm⁻¹

.

¹H NMR (500 MHz,

8232 | Org. Biomol. Chem., 2012, 10, 8216–8235

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CDCl₃): δ = 1.14–1.23 (m, 3 H), 1.32–1.40 (m, 2 H), 1.46 (s, 9

H), 1.58–1.64 (m, 1 H), 1.68–1.72 (m, 2 H), 1.87–1.95 (m, 4

H), 1.99–2.08 (m, 4 H), 2.26–2.43 (m, 4 H), 2.70–2.75 (m, 2

H), 3.47 (s, 2 H), 3.91–3.98 (m, 1 H), 4.17 (t, J = 6.9 Hz, 1 H),

4.26 (t, J = 7.5 Hz, 1 H), 4.36–4.47 (m, 2 H), 5.71 (br d, J = 8.0

Hz, 1 H), 6.94–7.03 (m, 3 H), 7.14 (d, J = 8.3 Hz, 2 H),

7.27–7.41 (m, 5 H), 7.57–7.59 (m, 4 H), 7.76 (d, J = 7.5 Hz,

2 H), 8.04 (br d, J = 7.4 Hz, 1 H), 8.67 (br s, 1 H). ¹³C NMR

and DEPT (125 MHz, CDCl₃): δ = 24.86 (2 CH₂), 25.55 (CH₂),

28.09 (3 CH₃), 30.28 (CH₂), 30.66 (2 CH₂), 32.88 (2 CH₂),

34.08 (CH₂), 47.26 (CH), 47.46 (2 CH₂), 51.68 (CH), 53.87

(CH), 61.96 (CH₂), 64.74 (C), 67.28 (CH₂), 83.07 (C), 114.78

(d, J = 20.7 Hz, CH), 117.26 (d, J = 21.9 Hz, CH), 119.88

(2 CH), 120.16 (2 CH), 120.19 (CH), 125.13 (CH), 125.21

(CH), 125.64 (CH), 127.21 (2 CH), 127.93 (2 CH), 129.92

(CH), 130.41 (d, J = 9.4 Hz, CH), 137.90 (d, J = 9.8 Hz, C),

138.18 (C), 141.42 (2 C), 143.63 (C), 143.87 (2 C), 157.08

(CvO), 162.99 (d, J = 247.8 Hz, C), 167.09 (CvN), 170.74

(CvO), 171.04 (CvO), 180.07 (CvO). ESI-MS: m/z = 857.8

[M + H]⁺.

1.98–2.09 (m, 4 H), 2.17–2.41 (m, 3 H), 2.68–2.77 (m, 3 H),

3.45–3.51 (m, 1 H), 3.60–3.69 (m, 2 H), 3.94–4.00 (m, 1 H),

4.14–4.30 (m, 2 H), 7.18–7.33 (m, 6 H), 7.39–7.49 (m, 4 H),

7.63 (t, J = 7.1 Hz, 2 H), 7.69–7.72 (m, 2 H), 7.89 (d, J =

7.6 Hz, 2 H), 8.14 (m, 1 H), 12.62 (br s, 1 H). HPLC ret. time =

14.9 min, 99.9%. ESI-MS: m/z = 801.7 [M + H]⁺.

50: A mixture of 48 (57.3 mg, 92.5 μmol), silica gel (0.570 g)

and toluene (15 mL) was stirred at reﬂux for 6 h. The reaction

mixture was diluted with CH₂Cl₂/methanol (10 : 1) and ﬁltered

through a short path of Celite®. After removal of the solvent 50

was obtained as a light yellow solid, yield: 44.8 mg (86%), m.p.

143–146 °C. IR (ATR): ν = 3261, 3051, 2926, 2854, 1700,

1587, 1530, 1490, 1402, 1336, 1249, 1134, 1092, 1072, 1005,

938, 893, 833, 767 cm⁻¹. HPLC ret. time = 6.6 min, 97.3%.

ESI-MS: m/z = 562.2 [M − H]⁻.

51: Compound 49 (51.4 mg, 64.2 μmol) was dissolved in

DMF (5 mL), piperidine (1 mL) was added and the reaction

mixture was stirred at room temperature for 3.5 h. After complete

removal of the solvent, the residue was washed twice with

diethyl ether (1 mL) and taken up in the minimal volume of

methanol. The product was precipitated by dropwise addition to

diethyl ether (100 mL), ﬁltered and dried. 51 was obtained as a

colourless solid, yield: 27.7 mg (75%), m.p. 178–180 °C. IR

(ATR): ν = 3316, 3064, 2930, 2856, 1693, 1600, 1531, 1515,

1473, 1451, 1410, 1366, 1338, 1308, 1249, 1155, 1080, 1051,

48: NaBH(OAc)₃(91 mg, 0.429 mmol) and portionwise 46

(63 mg, 0.216 mmol) were added to a solution of 42 (62.5 mg,

0.15 mmol) and EtN(i-Pr)₂(36 μL, 0.212 mmol) in 1,2-C₂H₄Cl₂

(2 mL). The reaction mixture was stirred for 19 h at room temp-

erature and then partitioned between Na₂CO₃(sat. aqueous) and

CH₂Cl₂. The organic layer was washed with H₂O, brine and

dried over Na₂SO₄. Removal of the solvent and puriﬁcation of

the residue by ﬂash chromatography (SiO₂; pentane/[CH₂Cl₂/

methanol 10 : 1], 20 : 1 to 1 : 1) afforded compound 48 as a col-

ourless solid, yield: 68.3 mg (73%), m.p. 102–104 °C. IR

(ATR): ν = 3263, 3183, 3114, 3058, 2930, 2855, 1723, 1692,

1585, 1530, 1491, 1455, 1396, 1367, 1339, 1308, 1241, 1139,

1006, 937, 892, 836, 776, 758, 739, 664 cm⁻^{1 1}H NMR

.

(500 MHz, CDCl₃/methanol-d₄, 3 : 1): δ = 1.03–1.26 (m, 6 H),

1.45–1.52 (m, 2 H), 1.56–1.64 (m, 3 H), 1.79 (br d, J = 10.1 Hz,

2 H), 1.89–2.07 (m, 8 H), 2.40–2.43 (m, 2 H), 2.64 (br s, 2 H),

3.15 (s, 2 H), 3.31 (br s, 2 H), 3.40 (t, J = 5.8 Hz, 1 H), 3.61 (br

s,1 H), 6.74 (dt, J = 9.5, 2.2 Hz, 1 H), 6.80 (d, J = 7.9 Hz, 1 H),

6.88–6.94 (m, 3 H), 7.15–7.20 (m, 1 H), 7.33 (d, J = 8.5 Hz,

2 H). HPLC ret. time = 3.2 min, 99.9%. ESI-MS: m/z = 579.5

[M + H]⁺.

1079, 1047, 957, 892, 824, 781, 756, 691 cm⁻^{1 1}H NMR

.

(500 MHz, CDCl₃): δ = 1.15–1.23 (m, 3 H), 1.32–1.40 (m,

2 H), 1.44 (s, 9 H), 1.61–1.67 (m, 1 H), 1.69–1.74 (m, 2 H),

1.95–2.10 (m, 8 H), 2.30–2.40 (m, 6 H), 2.73–2.78 (m, 2 H),

3.48 (s, 2 H), 3.90–3.96 (m, 1 H), 6.93–7.02 (m, 3 H), 7.15 (d,

J = 8.4 Hz, 2 H), 7.30–7.35 (m, 1 H), 7.52 (d, J = 8.3 Hz, 2 H),

7.99 (br s, 1 H), 8.09 (br d, J = 8.0 Hz, 1 H). ¹³C NMR and

DEPT (125 MHz, CDCl₃): δ = 21.17 (2 CH₂), 24.88 (CH₂),

25.57 (CH₂), 28.22 (3 CH₃), 30.72 (2 CH₂), 32.88 (2 CH₂),

34.58 (CH₂), 36.54 (CH₂), 47.48 (2 CH₂), 51.74 (CH), 61.94

(CH₂), 64.75 (C), 80.74 (C), 114.83 (d, J = 20.8 Hz, CH),

117.30 (d, J = 21.8 Hz, CH), 119.91 (2 CH), 125.69 (d, J =

2.7 Hz, CH), 129.92 (2 CH), 130.42 (d, J = 9.3 Hz, CH), 131.61

(C), 137.89 (d, J = 9.7 Hz, C), 138.04 (C), 162.98 (d, J =

247.4 Hz, C), 167.15 (CvN), 171.08 (CvO), 172.98 (CvO),

180.11 (CvO). ESI-MS: m/z = 620.9 [M + H]⁺.

Typical procedure for the preparation of the peptides

H-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-4Gl-3Gl-4Gl-3Gl-4Gl-Asp-

(ODhc)-NH₂(6a), where Sta is (3S,4S)-4-amino-3-hydroxy-6-

methylheptanoic acid. Loading and automated assembly of Asp

(ODhc)¹⁴to Glu¹: the PAL-PEG-PS resin (0.38 mmol g⁻¹

,

0.30 mmol) was subjected to automated deprotection, and

loaded with Fmoc-Asp(ODhc)-OH, which was automatically

activated and coupled. While the majority of the amino acids

was coupled using the standard cycle as described above, the

building blocks 4d–g, 24, 28, 32, 33, 41, 44, 49 and 50 were

manually activated: the amino acid (2 equiv. based on the resin)

was dissolved in DMF (2 mL). HATU (2 equiv. based on the

resin) and EtN(i-Pr)₂(2 equiv. based on the resin) were added

and the mixture was stirred at room temperature for 10 min.

Then EtN(i-Pr)₂(2 equiv. based on the resin) was added and the

mixture was transferred to the synthesiser, where coupling,

washing and deprotection took place. Cleavage and post-

cleavage processing: after washing with CH₂Cl₂and drying in

vacuo, the peptide was cleaved by stirring in CF₃COOH/

(i-Pr)₃SiH/H₂O (95 : 2.5 : 2.5) at room temperature for 90 min.

After removal of the resin by ﬁltration, the crude peptide was

collected by precipitation from pentane/diethyl ether (1 : 1) at

49: Triﬂuoroacetic acid (5 mL) was added slowly at 0 °C to a

solution of 47 (50.1 mg, 58.4 μmol) in CH₂Cl₂(5 mL). The

mixture was stirred at room temperature for 90 min, diluted with

CH₂Cl₂and washed four times with water and once with brine.

Drying over Na₂SO₄and removal of the solvent afforded 49 as a

light yellow solid, yield: 36.5 mg (78%), m.p. 156–159 °C. IR

(ATR): ν = 3325, 2930, 2857, 1694, 1662, 1601, 1576, 1529,

1473, 1450, 1411, 1369, 1338, 1249, 1155, 1081, 1055, 1007,

938, 835, 775, 758, 737, 664 cm⁻^{1 1}H NMR (500 MHz,

.

DMSO-d₆): δ = 1.07–1.16 (m, 1 H), 1.23–1.32 (m, 5 H), 1.61

(d, J = 12.0 Hz, 1 H), 1.69–1.80 (m, 3 H), 1.84–1.94 (m, 3 H),

Org. Biomol. Chem., 2012, 10, 8216–8235 | 8233

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−78 °C and subsequent centrifugation. The centrifugate was dis-

solved in methanol/acetonitrile/H₂O/AcOH (20 : 20 : 10 : 1). Pre-

parative HPLC with a gradient from 50% to 85% of eluent B in

20 min (product ret. time: 18.4 min) afforded after drying 6a as

a colourless lacquer, yield: 0.174 g (57%). HPLC ret. time =

27.5 min, 99.9%. ESI-MS: m/z = 2596.5 [M + H]⁺, 2614.4

[M + NH₄]⁺, 2619.5 [M + Na]⁺.

corresponding graphs represent data from at least two exper-

iments. For four other compounds (free pharmacophores 41, 17,

51 and anchor variant 6g), at least two experiments were con-

ducted over the full concentration range (nine inhibitor dilutions

and DMSO control), while in a third experiment a reduced

number of concentrations was used to conﬁrm the EC₅₀. After

4–5 h incubation at 37 °C and 5% CO₂, the cell culture super-

natants from 6 wells of a 96 well culture plate were pooled and

measured in duplicate with the Aβ40 ECL assay. The signals

were normalised to % of the DMSO controls. Means of the nor-

malised duplicate values were plotted against inhibitor concen-

tration and the log EC₅₀values were calculated by non-linear

regression using GraphPad Prism 5.01 software (GraphPad Soft-

ware Inc., La Jolla, USA).

Cell culture

An SH-SY5Y cell line stably overexpressing wild-type APP695

with an N-terminal Myc tag and a C-terminal Flag tag⁴⁶was

kindly provided by L. Münter and G. Multhaup (Freie Universi-

tät Berlin). The cells were cultured in D-MEM/F12, sup-

plemented with 10% fetal calf serum, 2 m^{M L}-glutamine, non-

essential amino acids and 50 μg mL⁻¹hygromycin B. Cultures

were grown at 37 °C, 5% CO₂.

Statistical analysis

Statistical analysis was performed with the GraphPad Prism Soft-

ware, version 5.01. The initial result obtained from curve ﬁtting

by non-linear regression is the log EC₅₀of the compound (not

the EC₅₀, which is calculated in a second step). To compare the

potency of different compounds, the respective log EC₅₀values

from at least 3 independent experiments were averaged and sub-

jected to statistical tests. For two compounds, Student’s t-test

(two-tailed, unpaired) was used to compare means and variations

of log EC₅₀values, while a one-way ANOVA was employed for

comparison of three or more compounds. In this case, either

Dunnett’s (comparison of all compounds to one reference com-

pound) or Tukey’s (pairwise comparison of all compounds)

post-test was added to correct for multiple testing. In our pre-

vious publication,²⁶we pooled normalised values from three

independent measurements to calculate the EC₅₀by non-linear

regression from the pooled dataset. This method leads to slightly

different EC₅₀values. In this study, pooled datasets were used

only for the representation of dose–response curves in graphs,

allowing a more clear visualisation. For all statistical analyses,

log EC₅₀means and standard deviations from three independent

measurements were used, because this method facilitates com-

parison of multiple compounds. A comparison of log EC₅₀and

de-logarithmised EC₅₀values calculated by averaging values

from three or more experiments with EC₅₀values obtained by

non-linear regression analysis of pooled datasets revealed very

small differences (ESI, Table S2†).

Inhibitor treatments

The test compounds were initially dissolved in DMSO at con-

centrations of 5 or 10 m^Mand stored in aliquots at −20 °C. For

the cell culture experiments, compound dilutions (100× of the

desired ﬁnal concentration in the test) were prepared in 100%

DMSO, and were freshly diluted 1 : 100 with cell culture

medium. The ﬁnal concentration of DMSO in the experiments

and controls was 1%. The culture medium in the cell culture

plates was replaced with the fresh medium containing the com-

pounds or 1% DMSO (blank test). For the ECL measurements

SH-SY5Y cells were seeded 24 h before treatment in 96 well

cell culture plates at a cell density of 2 × 10⁵cells cm⁻². The

compounds were diluted in the medium and added to the cells as

described above. For each condition six wells were used and

pooled after an incubation time of 4–5 h.

Electrochemiluminescence (ECL) assay

Aβ-peptides in cell supernatants were measured with the MSD®

Human (6E10) Abeta 40 Kit according to the manufacturer’s

protocol on a Sector™ Imager 6000 (Meso Scale Discovery).

The synthetic reference peptide dilutions were prepared in 1%

blocker A. The fetal calf serum present in the SH-SY5Y cell

culture medium contains bovine Aβ-peptides which are detected

by the ECL assay. The amount of the bovine Aβ40 in an uncon-

ditioned culture medium was determined to be ∼50 pg mL⁻¹

and this background value was subtracted from the calculated Aβ

,

Acknowledgements

concentrations in the SH-SY5Y cell culture experiments.

This work was supported by the FP7 EU grant NADINE (Grant

Agreement Number 246513) and by the grant PURE (Protein

Research Unit Ruhr within Europe) from the State Government

North Rhine-Westphalia. We thank H. Kamrowski-Kruck and

C. Janßen (University of Duisburg-Essen) for excellent technical

assistance and Dr. L. Münter and Prof. Dr. G. Multhaup (Free

University Berlin) for providing the stably transfected SH-SY5Y

cells. We are indebted to Dr. A. Shevchenko (MPI-CBG,

Dresden) for the measurement of the high resolution ESI mass

spectra. We are grateful to Dr. G. Jennings (JADO Technologies

GmbH, Dresden) for helpful discussions.

Dose–response curves and EC₅₀determinations

For the calculation of dose–response curves and EC₅₀values for

SH-SY5Y APPwt cells, all compounds were tested in three inde-

pendent experiments in approximately half-logarithmic serial

dilutions. For six compounds (6d, 6e, 7m, 16, 19 and 20),

ranges of inhibitor concentrations were altered in different exper-

iments in order to deﬁne both top and bottom plateaus of the

dose–response curve. Therefore, not each single concentration

was analysed in all three experiments. Error bars in the

8234 | Org. Biomol. Chem., 2012, 10, 8216–8235

View Article Online

24 T. Braxmeier, T. Friedrichson, W. Froehner, G. Jennings, M. Munick,

G. Schlechtingen, C. Schroeder, H.-J. Knölker, K. Simons, M. Zerial and

T. Kurzchalia, International Patent, WO 05/097199, 2005; PCT/EP2005/

003740, 2005.

25 L. Rajendran, A. Schneider, G. Schlechtingen, S. Weidlich, J. Ries,

T. Braxmeier, P. Schwille, J. B. Schulz, C. Schroeder, M. Simons,

G. Jennings, H.-J. Knölker and K. Simons, Science, 2008, 320, 520.

26 H. Schieb, S. Weidlich, G. Schlechtingen, P. Linning, G. Jennings,

M. Gruner, J. Wiltfang, H.-W. Klafki and H.-J. Knölker, Chem.–Eur. J.,

2010, 16, 14412.

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Article Doi

Optimisation of BACE1 inhibition of tripartite structures by modification of membrane anchors, spacers and pharmacophores - Development of potential agents for the treatment of Alzheimer's disease

DOI: 10.1039/c2ob26103k

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