Aggregation and supramolecular membrane interactions that influence anion transport in tryptophan-containing synthetic peptides

FULL PAPER

DOI: 10.1002/chem.201103203

Aggregation and Supramolecular Membrane Interactions that Influence

Anion Transport in Tryptophan-Containing Synthetic Peptides

Megan M. Daschbach,^[a]Saeedeh Negin,^[a]Lei You,^[a]Matthew Walsh,^[b]and

George W. Gokel*^{[a, c]}

Abstract: Self-assembly is a desired

property in supramolecular chemistry,

but extensive aggregation may be

counterproductive. Rigid systems typi-

cally have better organization, but are

inherently less dynamic. This work

shows that ion transport by amphiphilic

heptapeptides (synthetic anion trans-

porters or SATs) is affected by aggre-

gation of the monomers in the bulk

aqueous phase to which they are added

and within the bilayer. Ion transport

was assessed for all compounds by

assay of Cl^ꢀrelease from liposomes.

The mechanism of ion transport was

confirmed by planar bilayer conduc-

tance studies for two compounds at op-

posite ends of the efficacy scale. Dy-

namic light scattering, the Langmuir

trough, transmission electron microsco-

py, ion release from liposomes, and

planar bilayer conductance studies

were used to assess the importance of

self-assembly versus aggregation in ion

transport. Generally, greater aggrega-

tion was has an adverse effect on the

transport, although at least dimeriza-

tion is required for amphiphilic hepta-

peptides to readily transport Cl^ꢀ.

Anion transport in these systems was

found to be sensitive to changes in the

C-terminal portion of the (Gly)₃Pro-

AHCTUNGRTENN(GUN Gly)₃sequence. Moreover, a significant

difference in transport efficacy was ap-

parent when l-Trp was replaced by d-

Trp in the same position.

Keywords: aggregation

·

amphi-

philes · anions · peptides · synthesis ·

transport

Introduction

solution and in bilayer membranes using dynamic light scat-

tering (DLS), the Langmuir (Langmuir–Blodgett or LB)

trough, transmission electron microscopy (TEM), ion re-

lease from synthetic vesicles, and planar bilayer conductance

measurements.

Most of the synthetic, pore-forming amphiphiles reported

thus far were designed to be injected into the bulk aqueous

phase that surrounds either a liposome or a cell. The com-

pound must at least insert into the bilayer from the aqueous

phase and, depending on the design, may be required to as-

sociate within the bilayer. To be sure, some very simple

structures can form pores. The classic example is Triton X-

100,^[7]which showed ion transport activity in a planar bilayer

conductance experiment when embedded in the bilayer

during membrane formation. When added from the bulk

phase, channel function by this simple detergent was not ob-

served.^[8]

The challenge of recognizing, complexing, and transporting

anions^[1]such as chloride is currently a major subdiscipline

in the broader field of supramolecular chemistry. The impor-

tance of binding and transport in biology is evident from the

extensive work underway on chloride transporters.^[2]The

highly evolved natural transporters such as the ClC family

of proteins^[3]create ion pathways through the entire mem-

brane, but other configurations are possible that engender

chloride transport.^[4,5]During recent decades, a range of syn-

thetic transporters has been reported.^[6]The design of such

synthetic transporters is fraught with an inherent contradic-

tion. Stronger binding of ions is favorable for recognition,

but impedes transport. Likewise, if self-assembly is required

for function, extensive aggregation may be counterproduc-

tive. In this study, we record the behavior of synthetic anion

transporters (SATs) of the general form (C₁₈H₃₇)₂N-CO-

Amphiphiles added to an aqueous suspension of lipo-

somes may disperse or aggregate in water, they may parti-

tion into the bilayer, an aggregate in the bulk phase may

fuse with the liposomal bilayer, the monomers may aggre-

gate within the bilayer and aggregates may dissociate into

monomers or smaller aggregates. Each of these reactions is

part of the pore-formation dynamic, but relatively little sys-

tematic study has been devoted to this issue, notwithstand-

ing our own previous study of these possibilities with respect

CH₂OCH₂CO-(Gly)₃-Pro-(Aaa)₃-OACHTUNGRTNE(UNG CH₂)₆CH₃in aqueous

[a] M. M. Daschbach, S. Negin, L. You, G. W. Gokel

Center for Nanoscience, and

Departments of Chemistry & Biochemistry and Biology

University of Missouri—St. Louis, St. Louis, MO 6312 (USA)

E-mail: gokelg@umsl.edu

[b] M. Walsh

Department of Science and Mathematics

Maryville University, St. Louis, MO 63141 (USA)

to R¹N-COCH₂OCH₂CO-(Aaa)₇-OR²has addressed any of

2

these issues for synthetic amphiphiles.^[9]

[c] G. W. Gokel

The importance of such studies is exemplified by recent

Departments of Developmental Biology and Chemistry

Washington University, St. Louis, MO 63130 (USA)

reports by Yang and co-workers^[10]of an aromatic bis-

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ACHTUNGTRENNUNG(leucine) derivative that shows classic open–close, channel-

type behavior, but is far too small to span a bilayer. A self-

assembly mechanism has been reported by DeGrado and

co-workers for various lysine-substituted isophthalamides

that exhibit antibiotic activity.^[11]To our knowledge, the

mechanism of transport remains obscure or unreported in

both cases and, indeed, in most systems despite the fact that

self-assembly must be critical. It is certainly true that studies

have been reported in which insertion dynamics have been

addressed, but these did not consider aqueous solution ag-

gregation or aggregate insertion into the bilayer. The ques-

tion of monomers associating with a membrane before inser-

tion has been considered by Matile,^[12]Fyles,^[13]and their co-

workers in the context of pore formation and also in the

context of antibiotic activity. Shaiꢁs “carpet” mechanism,^[14]

for example, recognizes that a two- or multistep process

must occur in order to lead to membrane penetration.

In the work reported here, we have incorporated trypto-

phan^[15]in the heptapeptide sequence. Tryptophan is the

rarest of the 20 common amino acids,^[16]but occurs in pro-

teins such as the KcsA potassium channel exclusively near

the membrane–aqueous interfaces.^[17]Various model systems

have been designed to probe the role of tryptophan as

a membrane anchor,^[18–20]both experimentally^[21]and compu-

tationally.^[22]Work in our laboratory showed that indole

itself could function as an amphiphilic headgroup in the for-

mation of vesicles.^[23]We have studied the behavior of

a family of amphiphilic heptapeptides by using the range of

techniques noted above and found dramatic differences in

aggregation behavior both in the bulk phase and the bilayer

as well as significant differences in ion transport function.

fragment comprising the fourth to seventh amino acids was

likewise prepared by using standard conditions, esterified,

and then linked to the acylated N-terminal fragment. The

general structure of the compounds discussed is shown

above and the sequences are identified by the amino acids

in the 5–7 positions in Table 1.

Table 1. Peptide sequence in (n-C₁₈H₃₇)₂N-COCH₂OCH₂CO-(Aaa)₇-O-n-

C₇H₁₅and percent of total Cl^ꢀrelease from DOPC/DOPA (7:3) lipo-

somes measured at 1800 s.

Sequence

Cl [%]

Sequence

Cl [%]

1

2

3

4

5

6

7

GGGPGGG

GGGPEGG

GGGPFGG

GGGPWGG

GGGPwGG

GGGPGAG

GGGPGEG

35

15

20

10

21

50

20

8

9

GGGPGWG

40

70

45

25

60

30

GGGPGGE

GGGPGGEACTHUNGTRENNNUG

ACHTUNGTRENNUNG

10

11

12

13

14

GGGPGGKACTHUNGTRENNNUG

GGGPGGW

GGGPGGWAHCUTNGTRENNUNG

AHCTUNGTRENNUNG

Results

The tryptophan-containing synthetic anion transporters

Compounds studied: Fourteen amphiphilic heptapeptides

with the general formula (n-C₁₈H₃₇)₂NCOCH₂OCH₂CO-

(Gly)₃-Pro-(Aaa)₃-O-n-C₇H₁₅were prepared for this study.

These amphiphiles contain three key elements. The N-termi-

nal, twin n-octadecyl chains comprise a hydrophobic mem-

brane anchor. The C-terminal n-heptyl chain serves as a sec-

ondary membrane anchor as well as blunting the carboxyl

groupꢁs charge. The N-terminal hydrocarbon chains are

linked to the peptide through diglycolic acid as its diamide.

The heptapeptide comprises the polar headgroup in which

the first four amino acids are Gly-Gly-Gly-Pro. All 14 com-

pounds are identical except for the three C-terminal amino

acids. Each compound can be specified by naming these

(4: WGG; 5: wGG; 8: GWG; and 12: GGW) are the pri-

mary subject of this study. The syntheses of the other com-

pounds have previously been reported (see the Experimen-

tal Section). Scheme 1 (below) provides an example for the

synthetic route used to obtain WGG, wGG, GWG, and

GGW.^[13]Note that “w” denotes d-stereochemistry, whereas

“W” denotes the more common l-isomer.

Chloride transport—different amino acid sequences and

membrane compositions: Chloride transport mediated by

WGG, wGG, GWG, and GGW was determined by using

vesicles prepared from either 1,2-dioleoyl-sn-glycero-3-phos-

phocholine (DOPC) or from 7:3 (w/w) DOPC and 1,2-dio-

leoyl-sn-glycero-3-phosphate (DOPA, see the Experimental

Section of reference [24]). Briefly, a KCl buffer was trapped

within the vesicles, which were then suspended in K₂SO₄

buffer. Each compound was then added to the vesicle sus-

pension and insertion into the bilayer and pore-formation

were inferred by detecting chloride release. The emergence

of Cl^ꢀfrom the liposomes was recorded by using a chloride-

selective electrode inserted into the vesicle suspension.^[27]At

the end of each trial, sufficient detergent (Triton X-100) was

three

amino

acids.

Thus,

we

refer

to

(n-

C₁₈H₃₇)₂NCOCH₂OCH₂CO-(Gly)₃-Pro-Glu-Gly-Gly-O-n-

C₇H₁₅(2) as EGG (for Glu-Gly-Gly).

The amphiphilic heptapeptides were synthesized by using

wet chemical methods^[24]as follows. Dioctadecylamine was

heated in toluene with diglycolic anhydride to give (n-

C₁₈H₃₇)₂NCOCH₂O-CH₂COOH after evaporation of the sol-

vent. This fragment was coupled to commercial triglycine to

give (n-C₁₈H₃₇)₂NCOCH₂O-CH₂CO-(Gly)₃-OH. The peptide

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Anion Transport

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Scheme 1. Synthetic route to 12, GGW. N-terminus: a) 1-heptanol, toluene, 58%; b) 1. N-Boc-Gly, EDCI, HOBt, NEt₃, CH₂Cl₂, 62%, 2. Boc removed

with 4n HCl/dioxane; c) 1. N-Boc-Gly, EDCI, HOBt, NEt₃, CH₂Cl₂, 56%, 2. Boc removed with 4n HCl/dioxane; d) 1. N-Boc-Pro, EDCI, HOBt, NEt₃,

CH₂Cl₂, 98%, 2 HCl/dioxane. C-terminus (synthesis has been described previously^[25]): e) toluene, reflux, 87%; f) TsOHGly-Gly-Gly-OCH₂Ph, EDCI,

HOBt, NEt₃, CH₂Cl₂, 89%; g) H₂, 10% Pd/C, ethanol, 96%. Final Coupling: h) EDCI, HOBt, NEt₃, CH₂Cl₂, 38%; i) NaOH, THF, 73%.

added to induce vesicular lysis and the total chloride con-

centration was determined. The release data were normal-

ized to [Cl^ꢀ]_final. The chloride release data for WGG, wGG,

GWG, and GGW are shown in Figure 1. Each data set is the

average of at least three independent measurements.

Release of Cl^ꢀfrom vesicles mediated by WGG, wGG,

GWG, or GGW, is a well-behaved process. The 1800 s time

value was chosen because ion release has often reached

a plateau by that time and/or the trend in transport behavior

is clear.^[28]Several of the experiments were monitored to

longer times, but those data sets were truncated for consis-

tency of presentation. In no case was any anomaly observed

at longer times.

The difference in the abilities of WGG, wGG, GWG, and

GGW to transport Cl^ꢀis striking. The fractional ion release

values from DOPC/DOPA (7:3) liposomes for Cl^ꢀobserved

at the 1800 s time point are WGG=0.10, wGG=0.21,

GWG=0.40, and GGW=0.25. Thus, for these peptides,

transport diminishes in the order GWG>GGW>wGG>

WGG. The same general trend is observed for fractional ion

release from DOPC liposomes. At 1800 s, the normalized re-

lease values are as follows: WGG=0.28, GGW=0.16,

WGG=0.11, and wGG=0.08. Thus the trend in the absence

of DOPA is GWG>GGW>WGG>wGG.

The percentage of chloride released from DOPC/DOPA

(7:3) at 1800 s is summarized in Table 1 for compounds 1–

14. Each of these experiments was conducted as described

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G. W. Gokel et al.

pore formation. The studies reported here used asolectin

membranes and the buffers contained 450 mm KCl and

10 mm HEPES at pH 7. Each ionophore was dissolved in

2,2,2-trifluoroethanol (TFE). The solution was added to the

cis-chamber resulting in a compound concentration of 2 mm.

Fyles^[13]has pointed out that “[v]esicle techniques offer

more scope for structure–activity surveys, and there are

many examples of this type of investigation. Even so, vesicle

techniques are subject to artefacts as the hoped-for channel

activity is often indistinguishable from the detergent-like

lysis of the vesicles.” In the present study, the parent pep-

tide, GGGPGGG, and variants WGG, and wGG were ex-

amined by the planar bilayer conductance method. The last

two compounds are the poorest transporters among the

tryptophan-containing peptides. The channel-forming prop-

erties of GGG are well documented.^[26]Our assumption was

that if a good transporter (GGG) and the poorest Trp-con-

taining compounds (WGG, wGG) showed ion-channel be-

havior, it was reasonable to assume that the range of com-

pounds shown in Table 1 all formed conducting pores. In ad-

dition, previous studies of GGE (not shown in Table 1) in

DOPC/DOPA bilayers showed channel activity and a con-

ductance of about 30 picoSiemens (pS).^[27]All three pep-

tides, GGG, WGG, and wGG, showed well-defined open–

close behavior at applied potentials between 20 and 50 mV

(see Figure 2).

Figure 1. Chloride release from DOPC/DOPA (7:3) liposomes (0.31 mm,

left panel) and DOPC liposomes (0.31 mm, right panel) mediated by 4

(WGG), 5 (wGG), 8 (GWG), and 12 (GGW) (65 mm at pH 7). No Cl^ꢀ

leakage above baseline was observed in the absence of a transporter.

above for WGG, wGG, GWG, and GGW. The indoyl side

chain of tryptophan can form hydrogen bonds, cation–p con-

tacts, and it adds steric bulk to the SAT. To be sure that the

effect of tryptophan substitution is unique to tryptophan

and not to any one of the aforementioned properties, we

have included substitutions that can form electrostatic con-

tacts, have different electron-rich p systems, and add steric

bulk. There is no evident trend. Steric bulk, such as that in 3

(FGG) decreased transport efficacy while that in 10 (GGE-

A

ACHTUNGTRENN(UNG NBD)) increased transport efficacy.

GGWACHTUNGTRENNUNG(N-CHO)) and alkylation (14, GGWAHCUTNGTREN(NUNG N-CH₃)) in-

creased and decreased the SATꢁs ability to transport Cl^ꢀ, re-

spectively. Finally, the identity of the p system (3, FGG),

the electron density of the p system (13, GGW

ACHTUNGTRNE(NUNG N-CHO)

and 14, GGW(N-CH₃)), and the stereochemistry of the tryp-

ACHTUNGTRENNUNG

tophan side chain (4, WGG and 5, wGG) all had varying ef-

fects on anion transport.

Figure 2. Single-channel traces for a) GGG (1), 20 mV; b) GGG (1),

40 mV; c) WGG (12), 50 mV; d) wGG (5), 50 mV.

Planar bilayer studies: Release of Cl^ꢀfrom liposomes may

occur either by a carrier mechanism or by pore formation.

These mechanisms may be distinguished by using the planar

bilayer voltage clamp conductance technique. The experi-

ment uses two chambers (cuvettes) connected by a hole of

approximately 200 mm. When a bilayer forms in the orifice,

solutions in the two chambers are insulated from one anoth-

er. Addition of a pore-former allows ions present in one

chamber to pass through the membrane and to carry cur-

rent, which is detected at a specific voltage (voltage clamp)

as a function of time. No current is detected when the pore

is closed. The observation of open–close behavior confirms

Figure 2 shows planar bilayer conductance data obtained

in asolectin membranes for GGG (panels a and b), WGG

(panel c), and wGG (panel d). The traces for GGG were ob-

tained at 20 and 40 mV and their conductances (ca. 6.5 and

11 pA) correspond. The results for WGG and wGG were

significantly different from those obtained for GGG and

from each other. The current for WGG was about 1.2 pA

and for wGG it was nearly sixfold greater (i.e., ca. 8 pA),

each at 50 mV. When the three amphiphilic peptides were

compared, they showed the trend GGG>wGG>WGG, re-

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Anion Transport

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gardless of whether transport was measured by ion release

from liposomes or by planar bilayer conductance measure-

ments.

Dynamic light scattering and transmission electron micros-

copy: In the experiments described herein, the ionophore is

added as a solution in an appropriate solvent to the aqueous

phase that bathes the membrane. When a solution contain-

ing the ionophore is added to the aqueous buffer, it is likely

that the amphiphiles aggregate as occurs in the formation of

micelles. The formation of aggregates in aqueous suspension

can be detected by dynamic light scattering (DLS). The

DLS method uses the scattering of a laser beam that passes

through a solvent (water in all cases described here) to

detect and determine the size of the aggregates formed in

the suspension. Compounds GGG (control), WGG, wGG,

GWG, and GGW were suspended in water (30 mm) in an

effort to detect aggregates. No aggregation was apparent for

GGG at any concentration studied (5–100 mm); at concen-

trations >100 mm, GGG precipitated from water. Similar

treatment of GWG gave data that suggested aggregation,

but the experiments were not reproducible. We infer that

while GWG does self-assemble in aqueous suspension,

a range of aggregates forms: the DLS instrument reported

different polydispersities in separate experiments conducted

at identical concentrations. Thus, nine trials yielded average

smaller and larger diameters of (50ꢁ6) and (155ꢁ53) nm,

respectively.

DLS showed stable, bimodal distributions for WGG,

wGG, and GGW. All three compounds formed aggregates

about 50 nm (ca. 500 ꢂ) in diameter. The larger aggregates

showed hydrodynamic diameters of approximately 150, 160,

and 120 nm, respectively. The aggregate distributions for

WGG, wGG, and GGW were reproducible [averages (9

trials), WGG: (56ꢁ4), (151ꢁ8) nm; wGG: (54ꢁ6), (163ꢁ

11) nm; GGW: (53ꢁ3), (116ꢁ9) nm] and stable for>24 h

[(WGG)_nwas stable for>72 h]. We note that GGG and

GWG are superior as Cl^ꢀtransporters to WGG, wGG, and

GGW. Thus, poor anion transport appears to correlate with

stable aggregate formation (WGG, wGG, or GGW). We

were unable to detect stable aggregate formation for good

Cl^ꢀtransporters GGG and GWG.

Figure 3. Transmission electron micrographs of aggregates comprised of

compound 4, (C₁₈H₃₇)₂NCOCH₂OCH₂CO-GGGPWGG-OC₇H₁₅, on

a copper grid. Left panel: 200 nm single structure. Right panel: 30-70 nm

distribution.

Figure 3 shows an aggregate larger than recorded by DLS,

but the latter is a statistical average. The structure shown

could be at the outer edge of the distribution or it could

have undergone a “pancake” effect during sample prepara-

tion. In either event, it is important to note that WGG,

wGG, and GGW form aggregates that can be detected by

more than one analytical technique and the more active

transporter amphiphiles GGG and GWG do not.

Langmuir trough isotherm data: The Langmuir trough pro-

vides a means to assess the dynamic organization of amphi-

philes at the air–water interface. The Langmuir trough con-

sists of a “pan of water” and two lateral barriers that can be

moved towards each other to reduce the available surface

area. An amphiphile is spread on the aqueous surface, the

barriers are moved to compress the monolayer, and the re-

sulting forces are reported by a transducer called a Wilhelmy

plate. The data obtained give information about the organi-

zation of the amphiphile, its minimum size requirements,

and the overall stability of the system.

We previously reported reproducible phase transitions at

(176ꢁ6) and (68ꢁ4) nm for GGG_.^[30]Similar studies were

conducted as part of this work with WGG, wGG, GWG, and

GGW. The trough experiments were replicated as many as

12 times for each amphiphile in a failed effort to achieve re-

producibility. Although generally similar behavior was ob-

served for each compound in the various runs, significant

enough differences were observed in enough replicates to

preclude a valid discussion of the data. Since our principal

interest concerning the function of the SATs was their be-

havior in the presence of phospholipids, the amphiphiles

were co-spread with either 1,2-dioleoyl-sn-glycero-3-phos-

phocholine (DOPC) or 1,2-dioleoyl-sn-glycero-3-phosphate

(DOPA), the same lipids used for the Cl^ꢀrelease studies

(see above).

The formidable DLS method gives no structural informa-

tion. Transmission electron micrographs (TEMs) were there-

fore obtained for any samples that were stable enough to be

studied by this method. Samples were prepared by evapora-

tion and deposition onto carbon-coated copper grids. We

previously observed bimodal distributions for related iono-

phores that had C-terminal benzyl rather than n-heptyl

esters only by DLS, but could not confirm them by micros-

copy.^[30]

The right panel of TEM Figure 3 shows spherical aggre-

gates (ca. 30–70 nm) of WGG that correspond to the smaller

size in the bimodal distribution. TEM images were obtained

when a single glycine replaced l-tryptophan in the fifth posi-

tion of the heptapeptide sequence (GGG!WGG), but no

image could be obtained for GGG. The left panel of

Compounds WGG, wGG, GWG, and GGW and the phos-

pholipid DOPC were co-spread (1:1 mol:mol DOPC/SAT)

on the aqueous phase of a Langmuir trough. Compounds

WGG, wGG, GWG, and GGW were also co-spread with the

phospholipids DOPC and DOPA (7:3:10 mol:mol:mol

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G. W. Gokel et al.

DOPC/DOPA/SAT). The trough barriers were moved from

a surface area of about 200 ꢂ²at a rate of approximately

20 ꢂ²molecule^ꢀ1min^ꢀ1until collapse was observed. The re-

sulting isotherms are shown in Figure 4.

The presence of DOPA (plus DOPC) appears not to

affect monolayer formation when mixed with WGG; the

two isotherms are essentially superimposable (Figure 4, top

panel). The monolayer formed from DOPA/DOPC and

GGW differs modestly from that formed from GGW and

DOPC alone. In the presence of DOPA (and DOPC),

a shift to smaller molecular areas are observed for GGW.

This suggests a closer molecular packing in the mixture. The

most pronounced shift occurs when GWG is co-spread with

DOPA: the monolayerꢁs transition point and collapse are

approximately 50 and 20 ꢂ²smaller, respectively. Figure 4

also reveals an interesting stereochemical effect for the iso-

mers WGG and wGG. The isotherms for the d-isomer,

wGG, show smaller molecular areas in the presence of

DOPA than when WGG is present with DOPC/DOPA. In

the absence of DOPA, wGG and WGG show very similar

results. This is discussed in a later section.

Discussion

Chemical equilibria involved in pore formation: In principle,

the formation of a conducting pore in a bilayer must involve

several steps. When the ionophore is added to an aqueous

suspension of vesicles, it can disperse, aggregate, or associate

with the external membrane of the liposome. If aggregates

form, the ability of amphiphile monomers to associate with

the liposomes will be reduced and the formation of pores

will ultimately be slowed. Once the ionophores associate

with the liposomes, they must insert in the bilayer. Aggrega-

tion may occur within the bilayer that does not involve pore

formation. Individual monomers can diffuse laterally within

the membrane, ultimately organizing into a conducting pore.

Each of these processes will have a rate specific to the

compound and medium. The combination of rates will deter-

mine how effectively, if at all, a pore forms. In addition, the

transport rate may be affected by the presence of a charge

within the conducting pore, as we have observed when a glu-

tamic acid residue is present in the heptapeptide.^[13]

Scheme 2 shows the minimum equilibria involved in pore

Scheme 2. Chemical equilibria involved in SAT pore formation.

Figure 4. Surface pressure-area (p–A) isotherm data for co-spread mono-

layers,

DOPC:ACHTUNGTRENNUNG(C₁₈H₃₇)₂NCOCH₂OCH₂CO-(Gly)₃-Pro-(Aaa)₃-OC₇H₁₅

(1:1, mol:mol), solid lines. Transitions: WGG (4) and DOPC: 152, 80 ꢂ.

GGW (12) and DOPC: 133, 71 ꢂ². wGG (5) and DOPC: 152, 75 ꢂ²

formation within a vesicular membrane when an amphiphilic

ionophore is added to an aqueous liposome suspension. Ide-

ally, k₁, k₃, k₄, and k₅would all be large and the reverse

rates would all be small. The formation of inactive aggre-

gates will be favored by larger values of k₂and k₆.

.

GWG (8) and DOPC: 180, 90 ꢂ². DOPC/DOPA:

(C₁₈H₃₇)₂-

ACHTUNGTRENNUNGNCOCH₂OCH₂CO-(Gly)₃-Pro-(Aaa)₃-OC₇H₁₅. (7:3:10, mol:mol:mol),

dashed lines. Transitions: WGG (4) and DOPC/DOPA: 154, 76 ꢂ². wGG

(5) and DOPC/DOPA: 138, 71 ꢂ². GWG (8) and DOPC/DOPA: 130,

68 ꢂ². GGW (12) and DOPC/DOPA: 132, 62 ꢂ².

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Dynamic light scattering and transmission electron micros-

copy: We have recently reported the use of various fluores-

cent probes to examine the behavior of SATs in aqueous so-

lution and in the phospholipid bilayer. The key findings of

those studies were as follows. First, SATs aggregate in the

bulk aqueous phase. The observation of pyrene excimer for-

mation demonstrated this for an appropriately substituted

SAT amphiphile/ionophore. Both monomer and excimer

emission were observed when these derivatives were insert-

ed in liposomes. The fluorescent dye NBD (4-fluoro-7-nitro-

benz-2-oxa-1,3-diazole) is quenched in water, but less in hy-

drocarbon media. Using this difference in behavior, we de-

termined that about 35% of an NBD-substituted SAT parti-

tioned into the bilayer.^[15]Formation of aggregated pores

was confirmed by fluorescence resonance energy transfer

from a tryptophan-substituted SAT to a pyrenyl-SAT.

The goal of the present study was to understand the dy-

namics of self-assembly and to assess whether aggregation,

either in the bilayer or the bulk aqueous phase, correlated

to transport. In all of our studies, we add the ionophore to

a membrane-containing phase rather than pre-mixing it.

This is done to preclude the type of behavior observed for

Triton X-100 described in the Introduction. The dynamic

light scattering technique was used in the expectation that

aggregation behavior and transport efficacy could be corre-

lated. As noted above, GGG is one of the more active Cl^ꢀ

transporters studied. No aggregates could be detected in

aqueous suspension at any concentration between 10–

100 mm. At higher concentrations, the compound precipitat-

ed from solution. These experiments suggest an inverse rela-

tionship between aqueous phase aggregation and bilayer

transport.

A number of SAT derivatives not included in the present

studies showed bimodal distributions by DLS, but only the

larger aggregates could be detected by TEM. When any of

the three C-terminal glycine residues (positions 5, 6, or 7)

was replaced by tryptophan, aggregation was observed

(DLS). Poor Cl^ꢀtransporters WGG, wGG, and GGW

showed similar bimodal distributions when suspended in

water. In all cases, the aggregates were calculated by the in-

strumentꢁs internal software to be about 150 nm and about

50 nm in diameter. In the case of GGW and WGG, both

DLS and TEM confirm the presence of large and small as-

semblies. The DLS software assumes spherical aggregates,

a credible assumption based on the TEM results shown

above (Figure 3).

Within the group of compounds GGG, WGG, wGG,

GWG, and GGW, it appears that the ability to form and to

observe aggregates in aqueous suspension is inversely corre-

lated to Cl^ꢀtransport efficacy. This is true whether trypto-

phan at position 5 has l- (WGG) or d-stereochemistry

(wGG). We note that Cl^ꢀtransport by the latter is signifi-

cantly better than its stereoisomer (see further discussion

below).

Planar bilayer conductance: The traces shown in Figure 2

(above) clearly show classic open–close behavior for GGG,

WGG, and wGG. The observed conductance for GGG

(35% Cl^ꢀrelease) is 257 picoSiemens (pS) compared to

28 pS for WGG (10% Cl^ꢀrelease). Using Hilleꢁs equation

to estimate pore size from conductance values,^[31]the pore

diameters of GGG and WGG are about 10 and 4 ꢂ, respec-

tively. We have previously reported an estimated pore size

for GGG of approximately 8–9 ꢂ based on computational

and physical models and on the synthesis of pseudo-

dimers.^[32]A solvated Cl^ꢀion is estimated to be a sphere of

about 6.5 ꢂ in diameter.^[33]The estimated pore size of ap-

proximately 4 ꢂ for WGG suggests that either it functions

as a monomer or that the dimer, if formed, is far more com-

pressed than for GGG. A smaller or more rigid pore would

certainly show poorer ion transport and this comports with

the poorer Cl^ꢀrelease measured for WGG in liposomal bi-

layers. Figure 2 also shows a significant difference in the be-

havior of WGG and wGG. The principal conductance state

of wGG is about sixfold larger than WGG. Since these two

molecules are isomers, the variance in conductance must

result from the difference in stereochemistry, that is, the ori-

entation of indolylmethyl group with respect to the main

peptide chain. In the liposomal studies, this difference is

only twofold, but it is in the same direction.

The kinetics of pore formation is also different for GGG

and WGG as judged from ion release experiments. Pore for-

mation and ion conductance are both observed first for

WGG rather than for GGG. However, GGG forms pores

that are larger and exhibit nearly tenfold greater conduc-

tance. The DLS data (see above) suggest that GGG does

not aggregate in an aqueous suspension, but WGG does. We

interpret this to mean that WGG aggregates contact the li-

AHCTUNGTREGpNNNU osomal surface and penetrate the bilayer as an aggregate.

Partial dissociation of monomers gives a conducting pore. In

contrast, the non-aggregated GGG monomers likely insert

individually into the bilayer and must then diffuse within

the membrane and organize into a pore. Since the pore

formed from GGG is larger than that formed from WGG, it

must require at least two monomers. Thus, ion currents are

observed for GGG more slowly than for WGG, but the con-

ductances are higher for the former once ions flow.

It is important to note that the higher conductance ob-

served by planar bilayer measurements for GGG than for

WGG corresponds to the vesicle-release data. This is so

even though ion release from vesicles is generally thought to

reflect insertion dynamics rather than the formation of an

Tryptophan-containing SAT GWG shows Cl^ꢀtransport

behavior (40% release) approximately equal to that of

GGG (35%) and superior to WGG (10%), wGG (21%), or

GGW (25%). DLS experiments indicated that GWG

formed aggregates in water. Unfortunately, neither size nor

polydispersity of these aggregates proved to be reproducible

in more than ten experiments. This behavior contrasts with

the aggregates formed from GGW, WGG, or wGG, which

formed well-defined aggregates that were stable in suspen-

sion for many hours.

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Table 2. Effect of lipid composition on monolayer formation and comparison with Cl^ꢀtransport rates.

Cl^ꢀrelease

[%]^[a]

Molecular areas [ꢂ²]

SAT+DOPC+ DOPA

Cl^ꢀrelease

[%]^[b]

Molecular area [ꢂ²]

SAT+DOPC

Change in Cl^ꢀ

release [%]

Molecular area [ꢂ²]

average change

WGG

wGG

GWG

GGW

10

21

40

20

154

138

130

131

76

71

68

62

10

28

16

152

180

133

80

75

90

71

0

11

12

4

1

9

35

5

[a] Cl^ꢀrelease from DOPC/ DOPA (7:3) vesicles. [b] Cl^ꢀrelease from DOPC vesicles.

ideal pore. The present study shows that the dynamic is

more complex.

to WGG means that the amino acid lies on opposite sides of

the main chain.

Langmuir trough studies of tryptophan-containing SATs:

Figure 4 shows the monolayer behavior of WGG, wGG,

GWG, and GGW in the presence of DOPC and DOPC/

DOPA; the molar ratios of which were 1:1 (SAT/DOPC)

and 10:7:3 (SAT/DOPC/DOPA). For the DOPC/DOPA sys-

tems, the phospholipid monomers are in the same ratio used

in the vesicle release experiments and in essentially all pre-

vious studies of these compounds. A molar equivalent of

SAT was added so that any organizational effect would be

apparent. In the vesicle experiments, the ratio of SAT to

phospholipids is lower (see Experimental Section). It is im-

portant to note that the reproducibility of the Langmuir

trough data is high. Each trace shown herein results from

a minimum of 9 and as many as 12 replicates. A difference

of 10% in a transition area or collapse pressure is signifi-

cant. Table 2 presents comparative data for Cl^ꢀrelease from

vesicles and the behavior observed when studied on the

Langmuir trough.

The second column of Table 2 summarizes the ion release

(as a percent, observed at 1800 s) from DOPC/DOPA (7:3)

vesicles. The order of transport efficacy is WGG<wGGꢂ

GGW<GWG. When the liposomes are comprised of

DOPC only, Cl^ꢀtransport is diminished overall, but the effi-

cacy order remains similar: WGGꢂwGG<GGW<GWG.

Table 2 also records the difference in percentage Cl^ꢀrelease

from liposomes and the average change in molecular area.

This gives a number that does not have independent physi-

cal meaning, but reflects organization in the monolayer and

can be correlated to the differences in transport values.

The presence of tryptophan in position 5, 6, or 7 of the

heptapeptide sequence clearly affects the interactions be-

tween the SAT and the surrounding phospholipids. The

monolayer behavior of WGG is the same in DOPC or in

DOPC/DOPA. The molecular areas of wGG, GWG, and

GGW are all shifted to smaller values in the presence of

DOPA. The magnitude of the change increases in the order

GGW<wGG<GWG. Chloride ion transport is also affect-

ed by the presence of DOPA, except for WGG (ca. 10% in

either lipid). For GGW, wGG, and GWG, fractional release

at 1800 s decreases in the absence of DOPA. The magnitude

of this change increases in the order GGW<wGG<GWG.

These data, like the aggregation data, suggest that molecular

organization and transport efficacy are correlated. Of

course, inversion of the stereochemistry in wGG compared

The effects of tryptophan: The presence of tryptophan may

alter the system in at least three different ways. First, trypto-

phan is regarded as a “membrane-anchoring group.” For ex-

ample, the only tryptophan residues that occur in the KcsA

potassium selective channel protein occur at the membrane

boundaries.^[34]Even the simple dimeric gramicidin chan-

nel^[35]appears to be anchored by tryptophans that occur

only at its proximal and distal boundaries.^[36]Other evidence

includes the formation of stable vesicles from monoalkyl-

substituted indoles.^[37]

Second, tryptophanꢁs indole residue has been extensively

studied as a donor in cation–p interactions that involve met-

allic and ammonium cations. Evidence for the latter

emerged a quarter century ago in the pioneering study of

peptide interactions reported by Burley and Petsko.^[38]

Dougherty and co-workers have demonstrated that cation–p

interactions involving Na⁺or K⁺can play a critical role in

channel function.^[39]Third, we have shown that synthetic ion

transporters show varying efficacies when cation–p interac-

tions between DOPC and aromatic side chains can occur.^[40]

These effects are not observed when DOPC is mixed with

DOPA, which helps to neutralize the formerꢁs ammonium

headgroup charge.

Postulated interaction between SAT and DOPA: It is well

established from our earlier work that the SAT amphiphiles

complex and transport Cl^ꢀions through the bilayer. An

NMR structure for a GGGPGGG peptide in the presence

of Bu₄N⁺Cl^ꢀwas obtained in collaboration with Tomich and

co-workers.^[41]The structure showed that Cl^ꢀwas bound by

the amide hydrogens of glycines 5 and 7. In principle, the

same general type of assembly may form on the Langmuir

trough in which an oxygen atom of DOPA acts as the anion

and the DOPC headgroup is positioned as is Bu₄N⁺in the

known complex. This possibility is illustrated in Figure 5,

both schematically and with space-filling models. Compound

wGG is positioned so that the DOPA headgroup resides in

the cavity known to interact with Cl^ꢀin the published

study.^[40]

As noted above (Figure 4), there is no difference in the

isotherms observed for WGG and DOPC in the presence or

absence of DOPA. This might seem unreasonable consider-

ing that DOPA is negatively charged and DOPC is neutral

(zwitterionic). Figure 6 shows an end view of diacetylphos-

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Anion Transport

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residue will be on one side of the extended molecule and on

the other when in position 6. Looking down on the structure

fragment shown in Scheme 3, positions 5 and 7 place the

side chains “upward” and position 6 faces “downward.” Re-

versing the stereochemistry from l to d in any position in-

verts this placement.

Scheme 3. C-terminal peptide sequence.

Stereochemistry, and thus chain orientation, may permit

or disallow the supramolecular interactions necessary for

the Trp moiety to function as an anchor or a cation–p

donor. Calculations and CPK molecular models (not shown)

suggest that the tryptophan is more exposed in wGG and

GWG than their isomers WGG and GGW when DOPA is

bound in the SATꢁs cleft. Thus, the indole side chains of

wGG and GWG could engage in more supramolecular con-

tacts. A favorable cation–p contact with a DOPC head-

group, while DOPA is bound in the SAT cleft, for example,

could account for the tighter packing at the air–water inter-

face in wGG and GWG (i.e., the shift to smaller molecular

areas in the SAT/DOPC/DOPA co-spread systems, see

Figure 4, above). The presumption that DOPA is organizing

the SAT and changing how the indole side chain is exposed

is also supported by the data shown in Figure 7.

Figure 5. Schematic and space-filling molecular model representations of

postulated DOPA-SAT interactions.

We prepared wGG, to assess if only the stereochemistry

of tryptophan (in WGG) could affect transport. The differ-

ence between wGG and WGG was confirmed in studies in-

volving ion release from vesicles (Table 2). We co-spread

wGG and WGG with DOPC alone and separately with

a mixture of DOPC/DOPA in a molar ratio of 7:3. No dif-

ference was observed in the monolayer behavior of WGG in

the presence of one or both lipids. The behavior of wGG

was significantly different: the molecular areas were an

average of 11 ꢂ²larger when wGG was in the presence of

DOPC/DOPA compared to when DOPA was absent. These

differences are apparent in the data shown in Figure 4

(above). Interestingly, in the absence of DOPA, the iso-

therms of wGG and WGG are essentially superimposed

(Figure 7).

These data indicate that when DOPA is absent, and there

is no presumed preorganization of the SAT cleft, the trypto-

phan would be equally exposed in the wGG and WGG sys-

tems. This presumably causes the monolayer packing to be

the same for either isomer. Indeed, this is confirmed by the

superimposed isotherm data sets for wGG and WGG in the

absence of DOPA shown in Figure 7.

Figure 6. Calculated space-filling molecular models showing the head-

groups (side and end views) of diacetylphosphatidylcholine (hydrogen

atoms omitted).

phatidylcholine. It is apparent that the three methyl groups

of choline essentially eclipse the phosphoryl oxygen atoms.

Thus, the size of choline or phosphate, when either is the

lipidꢁs terminal residue, is similar. Molecular models suggest

that either DOPA or DOPC can reside in the SATꢁs cleft, as

illustrated for DOPA in Figure 5. The complexation interac-

tion is expected to be enhanced for DOPA by electrostatics,

although the extent of the increase is hard to judge consider-

ing the multiplicity of weak forces that can act in this

system.

Additional confirmation can be found in the planar bilay-

er conductance results. In previous studies, the pores formed

from SATs were typically, approximately dimeric.^[32]If we

assume that is the case here, then differences in conductance

must relate to either the organization of the pore or to inter-

The space-filling representation in Figure 5 shows that

tryptophan protrudes from the peptide chain and the direc-

tion of that protrusion is dependent on the amino acidꢁs ste-

reochemistry. If a side chain is present at position 5 or 7, the

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G. W. Gokel et al.

were shown to form pores. Aggregation in aqueous suspen-

sion was assayed by dynamic light scattering. The proclivity

to aggregate in suspension was correlated to ion release

from liposomes. Aggregation was further confirmed by

transmission electron microscopy. The general conclusion

from these studies was that those compounds that self-ag-

gregated were generally poor ion transporters.

Tryptophan (Trp, W) was introduced as a replacement for

glycine on the C-terminal side of proline in the heptapeptide

sequence GGGPGGG. The presence of W in positions 5 or

7 gave poorer Cl^ꢀtransport activity than when the triglycine

sequence was unperturbed. Good Cl^ꢀtransport was ob-

served when W was present at position 6. Differences in Cl^ꢀ

transport were also observed for the replacement of l-Trp

with d-Trp in position 5. This was attributed to differences

in steric interactions resulting from the side chain orienta-

tion in a presumed linear (sheet) conformation. Analyses

using the Langmuir trough confirmed that better organiza-

tion (closer packing) in the monolayer correlated to poorer

ion transport. Differences in membrane composition

(DOPC vs. DOPC/DOPA) suggested that the SAT mole-

cules can interact with and bind the phosphoryl headgroup

of DOPA, thus affecting molecular organization and pore

formation.

Figure 7. Surface pressure-area (p–A) isotherm data for co-spread mono-

layers,

DOPC:ACHTUNGTRENNUNG(C₁₈H₃₇)₂NCOCH₂OCH₂CO-(Gly)₃-Pro-(Aaa)₃-OC₇H₁₅

(1:1, mol:mol). Grey trace: WGG (4) and DOPC, Transitions: 152, 80 ꢂ².

Black trace: wGG (5) and DOPC, Transitions: 152, 75 ꢂ²

.

nal hindrance. In a study of GGE,^[29]we found that the pres-

ence of ionizable glutamate within the pore decreased ion

transport. This was ascribed to the negative charge impeding

Cl^ꢀtransport within the dimeric pore. In the present case,

tryptophan may play one of two evident roles. On the one

hand, it may turn outward from the pore and interact with

positive lipid headgroups in a cation–p fashion. This could

give a higher level of organization to the pore and enhance

transport. Alternately, it may infiltrate the pore and reduce

transport simply by steric hindrance.

Experimental Section

Three different types of information are available with re-

spect to this issue. First, we have measured ion release,

which reflects insertion and pore-formation dynamics.

Second, we have measured planar bilayer conductance,

which reflects the effectiveness of transport within a pore.

Third, we have information from the Langmuir trough con-

cerning organization of the peptides both in the absence and

presence of lipids. Chloride ion release from liposomes is

mediated more effectively by wGG than by WGG (see

Table 1). Planar bilayer conductance shows that wGG is

a better pore-forming transporter than is WGG (Figure 2).

Figure 4 shows that wGG, but not WGG, is sensitive to the

presence of DOPA, suggesting a higher level of organization

within the bilayer. In the absence of DOPA, d-tryptophan

impedes the transport pathway less than does l-Trp. We

conclude that the steric position of tryptophan, dictated by

the two possible stereochemical arrangements, is critical to

interactions with the anionic headgroups of DOPA mono-

mers, which leads to a more organized conductance state.

General: ¹H NMR (300 MHz) and ¹³C NMR (125 MHz) spectra were re-

corded in CDCl₃unless otherwise specified and are reported as follows:

chemical shifts reported in ppm (d) downfield from internal (CH₃)₄Si (in-

tegrated intensity, multiplicity (br=broad, s=singlet, d=doublet, t=trip-

let, q=quartet, brs=broad singlet, m=multiplet, etc.), coupling con-

stants in Hz, assignment. ¹³C NMR spectra are referenced to CDCl₃

(77.23 ppm). Infrared spectra were recorded on a Perkin–Elmer 1710

Fourier transform infrared spectrometer. Melting points were determined

on a Thomas Hoover apparatus in open capillaries and are uncorrected.

Thin-layer chromatography was performed on silica gel 60-F-254 with

a 0.2 mm thickness. Preparative chromatography columns were packed

with silica gel (Merck grade 9385, 230–400 mesh, 60 ꢂ). Reagents were

of the best grade commercially available and were distilled, recrystal-

lized, or used without further purification, as appropriate. CH₂Cl₂was

distilled from calcium hydride. Reactions were conducted under N₂

unless otherwise noted. The following abbreviations are used throughout:

EDCI: 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride;

HOBt: 1-hydroxybenzotriazole; TsOH: toluenesulfonic acid; DGA rep-

ꢀ

resents diglycoyl, COCH₂OCH₂CO . Combustion analyses were per-

formed by M-HW Laboratories, Phoenix, AZ, and are reported as per-

centages.

Compound 1: (C₁₈H₃₇)₂NCOCH₂OCH₂CO-Gly-Gly-Gly-Pro-Gly-Gly-

Gly-OC₇H₁₅(1) was prepared as previously reported.^[24]

Compound 2: (C₁₈H₃₇)₂NCOCH₂OCH₂CO-Gly-Gly-Gly-Pro-Glu-Gly-

Gly-OC₇H₁₅(2) was prepared as previously reported.^[29]

Conclusion

Compound 3

ACHNUTGTER(NGNUN C₁₈H₃₇)₂NCOCH₂OCH₂CO-Gly-Gly-Gly-Pro-OCH₂Ph: l-Proline benzyl

The work presented here addresses the relationship of self-

assembly and aggregation to the efficacy of Cl^ꢀtransport. A

range of compounds was prepared and their ability to trans-

port Cl^ꢀthrough liposomal membranes was confirmed.

Transporters at opposite ends of the efficacy scale were

tested by the planar bilayer conductance method and both

ester·HCl (0.47 g, 1.93 mmol) was dissolved in dry CH₂Cl₂and cooled to

08C. (C₁₈H₃₇)₂NCOCH₂OCH₂CO-Gly-Gly-Gly-OH (1.56 g, 1.93 mmol,

synthesis see reference [24]), EDCI (0.41 g, 2.12 mmol, 1.1 equivalents),

HOBt (0.29 g, 2.12 mmol, 1.1 equiv), and NEt₃(0.59 g, 5.79 mmol,

3 equiv, dropwise) were added successively to this solution. The reaction

was stirred at 08C for 30 min under N₂, warmed to RT and stirred for

48 h. The solvent was evaporated in vacuo and the resulting yellow oil

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Anion Transport

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was subjected to chromatography (silica gel, 95:5 CH₂Cl₂/MeOH) to

0.38 mmol; for synthesis see reference [24]), EDCI (0.08 g, 0.42 mmol),

HOBt (0.06 g, 0.42 mmol), and NEt₃(0.12 g, 1.14 mmol) were added. The

reaction was stirred (08C for 30 min, 48 h at RT). The solvent was evapo-

rated in vacuo, the resulting yellow oil was subjected to chromatography

(silica gel, 93:7 CH₂Cl₂/MeOH), and the product crystallized from

MeOH to afford a white solid (0.46 g, 95%). M.p. 118–1208C; ¹H NMR:

d=0.88 (t, J=6.9 Hz, 9H; CH₂CH₃), 1.25 (brs, 68H; NCH₂CH₂-

afford a white solid (1.82 g, 95%). M.p. 54–568C; ¹H NMR: d=0.87 (t,

J=6.9 Hz, 6H; CH₂CH₃), 1.31 (brs, 60H; NCH₂CH

(brs, 4H; NCH₂CH₂A(CH₂)₁₅CH₃), 1.98–2.01 (m, 4H; Pro NCH₂CH₂CH₂),

3.04 (t, J=8.1 Hz, 2H; NCH₂CH₂A(CH₂)₁₅CH₃), 3.25 (t, J=8.1 Hz, 2H;

NCH₂CH₂A(CH₂)₁₅CH₃), 3.49–3.61 (m, 2H; Pro NCH₂CH₂CH₂), 3.93–4.13

₂ACHTUGNERTN(NUNG CH₂)₁₅CH₃), 1.49

CHTUNGTRENNUNG

CTHUNGTRENNUNG

CHTUNGTRENNUNG

(m, 7H; Gly CH₂, Pro CH), 4.27 (s, 2H; COCH₂O), 5.14 (s, 2H;

OCH₂C₆H₅) 7.09 (t, J=4.5 Hz, 1H; NH), 7.28–7.38 (m, 5H; CH₂C₆H₅),

7.75 (t, J=5.7 Hz, 1H; NH), 8.31 ppm (t, J=6.1 Hz, 1H; NH); ¹³C NMR

(CDCl₃): d=14.3, 22.9, 24.8, 27.1, 27.2, 27.8, 29.0, 29.1, 29.5, 29.5, 29.6,

29.7, 29.8, 29.9, 31.5, 32.1, 42.0, 43.0, 46.2, 46.6, 47.0, 59.0, 59.3, 67.1, 67.8,

69.8, 72.0, 123.3, 128.5, 128.8, 128.9, 135.7, 167.2, 167.5, 168.7, 169.3,

170.0, 171.1, 171.8 ppm.

A

(CH₂)₄CH₃), 1.50–1.62 (m, 6H; NCH₂CH₂-

₂A(CH₂)₄CH₃), 1.65–2.06 (m, 4H; Pro

₂A(CH₂)₁₅CH₃, Pro CH), 3.28–

₂A(CH₂)₄CH₃), 3.69–3.80 (m,

E

CHTUNGTRENNUNG

CTHUNGTRENNUNG

N

CHTUNGTRENNUNG

AHCTUNGTRENNUNG

Gly CH_2,COCH₂O), 4.30 (s, 2H; COCH₂O), 4.55 (m, 1H; Phe NHCH),

7.09 (brs, 1H; NH), 7.20–7.32 (m, 5H; Phe C₆H₅), 7.41 (t, J=5.0 Hz, 1H;

NH), 7.55 (t, J=6.0 Hz, 1H; NH), 7.75 (t, J=5.0 Hz, 1H; NH), 7.83 (t,

J=6.2 Hz, 1H; NH), 8.55 ppm (brs, 1H; NH); ¹³C NMR: d=14.3, 22.8,

22.9, 24.6, 26.0, 27.1, 27.3, 27.9, 28.7, 29.1, 29.3, 29.5, 29.6, 29.8, 29.8, 29.9,

31.9, 32.1, 36.2, 41.5, 42.6, 43.1, 43.7, 46.6, 41.2, 47.7, 55.1, 61.7, 65.9, 70.0,

72.1, 77.4, 126.8, 128.6, 129.0, 129.4, 138.2, 168.7, 169.8, 170.2, 170.4,

170.6, 171.0, 171.6, 172.0, 172.0, 172.5 ppm; ES-MS: m/z (%): 1265.9

(100).

ACHTUNGTRENNUNG

(1.80 g,

1.81 mmol, 1.0 equiv) was dissolved in hot EtOH (80 mL), 10% Pd/C

(0.55 g) was added (Parr shaker), and the suspension was shaken (60 psi

H₂, 3 h, RT, monitored by TLC 95:5 CH₂Cl₂/MeOH). The mixture was

filtered (celite) and evaporated in vacuo to give white solid (1.69 g,

99%). ¹H NMR: d=0.87 (t, J=7.0 Hz, 6H; CH₂CH₃), 1.25 (brs, 60H;

NCH₂CH

(m, 4H; Pro NCH₂CH₂CH₂), 3.09 (t, J=7.4 Hz, 2H; NCH₂CH₂-

(CH₂)₁₅CH₃), 3.27 (t, J=7.5 Hz, 2H; NCH₂CH₂A(CH₂)₁₅CH₃), 3.52–3.73

₂ACHTUNGTRENNUNG(CH₂)₁₅CH₃), 1.51 (brs, 4H; NCH₂CH₂CAHTUGNTERN(NUGN CH₂)₁₅CH₃), 1.98–2.22

Compound 4

A

CHTUNGTRENNUNG

Boc-WACHTUNGTRNNEUG(N-CHO)GG-OC₇H₁₅: Boc-WCAHTUNGTRENN(UGN N-CHO)-OH (0.50 g, 1.50 mmol),

glycylglycine n-heptyl ester tosylate^[29](0.61 g, 1.50 mmol), EDCI (0.32 g,

1.66 mmol) and HOBt (0.22 g, 1.66 mmol) were dissolved in CH₂Cl₂

(30 mL), cooled at 08C, and Et₃N (0.63 mL) was added. The mixture was

stirred (24 h, RT), the solvent was evaporated in vacuo, the residue was

subjected to chromatography (silica gel, CHCl₃/MeOH=98:2) to afford

an oil (0.55 g, 67%). ¹H NMR: d=0.88 (t, J=6.8 Hz, 3H; CH₂CH₃),

(m, 2H; Pro NCH₂CH₂CH₂), 3.94–4.39 (m, 9H; Gly CH₂, Pro CH,

COCH₂O), 7.75 (brs, 1H; NH), 8.00 (brs, 1H; NH), 8.24 ppm (brs, 1H;

NH); ¹³C NMR: d=14.3, 22.9, 25.1, 27.1, 27.3, 27.8, 29.0, 29.5, 29.6, 29.8,

29.9, 29.9, 32.1, 42.0, 43.1, 46.7, 47.3, 60.0, 69.4, 71.3, 168.3, 169.1, 170.1,

170.4, 171.4, 173.9 ppm.

ACHTUNGTRENNUNG(C₁₈H₃₇)₂NCOCH₂OCH₂CO-Gly-Gly-Gly-Pro-Phe-OBz: l-Phenylalanine

benzyl ester tosylate salt (0.24 g, 0.55 mmol, 1 equiv) was dissolved in dry

CH₂Cl₂and cooled to 08C under N₂. (C₁₈H₃₇)₂NCOCH₂OCH₂CO-Gly-

Gly-Gly-Pro-OH (0.50 g, 0.55 mmol), EDCI (0.12 g, 0.61 mmol), HOBt

(0.08 g, 0.61 mmol) and NEt₃(dropwise, 0.17 g, 1.66 mmol) was added to

this solution. The reaction was stirred (08C, 30 min then RT 48 h). The

solvent was evaporated in vacuo and the resulting yellow oil was subject-

ed to chromatography (silica gel, 97:3 CH₂Cl₂/MeOH) to afford a white

solid (0.50 g, 78%). ¹H NMR: d=0.88 (t, J=6.9 Hz, 6H; CH₂CH₃), 1.25

1.20–1.33 (m, 8H; OCH₂CH

(quint, J=7.0 Hz, 2H; OCH₂CH

CH₂), 3.85–4.05 (m, 4H; Gly CH₂), 4.10 (t, J=6.9 Hz, 2H; OCH₂CH₂-

G

ACHTUGNTRNEN(UNG CH₃)₃), 1.62

AHCTNUGTREN(GUNN CH₂)₄CH₃), 4.52 (brs, 1H; Trp CH), 5.44 (brs, 1H; NH), 6.90–9.10 ppm

(m, 8H; CONH, indole CH and CHO); ¹³C NMR: d=14.2, 22.7, 25.9,

28.0, 28.4, 28.7, 29.0, 31.9, 41.4, 43.2, 54.7, 66.0, 80.8, 116.4, 119.2, 124.0,

124.8, 125.7, 155.9, 159.7, 169.1, 170.0, 172.2 ppm.

Boc-PW

(N-CHO)GG-OC₇H₁₅

:

Boc-WACHNUTGTRENNUNG

(brs, 60H; NCH₂CH

1.79–1.82 (m, 4H; Pro NCH₂CH₂CH₂), 2.96–3.41 (m, 6H; NCH₂CH₂-

(CH₂)₁₅CH₃, Phe NHCH(CH₂C₆H₅)CO), 3.93–4.13 (m, 8H; Gly CH₂,

₂ACHTUNGTRENNUNG(CH₂)₁₅CH₃), 1.49 (brs, 4H; NCH₂CH₂AHCTUNTGRENN(GUN CH₂)₁₅CH₃),

0.97 mmol) was deprotected in 4n HCl/dioxane (RT, 2 h). HCl·WACHTUNGTRENNUNG

CHO)GG-OC₇H₁₅, Boc-Pro-OH (0.21 g, 0.97 mmol), EDCI (0.20 g,

1.07 mmol) and HOBt (0.14 g, 1.07 mmol) were dissolved in CH₂Cl₂

(30 mL), cooled at 08C, Et₃N (0.41 mL) was added, and the mixture was

warmed to RT (48 h). The solvent was evaporated in vacuo and the resi-

due was subjected to chromatography (silica gel, CHCl₃/MeOH=98:2) to

give a white solid (0.44 g, 70%). ¹H NMR: d=0.88 (t, J=6.7 Hz, 3H;

A

ACHTUNGTRENNUNG

COCH₂O), 4.30 (s, 2H; COCH₂O), 4.52 (m, 2H; Phe NHCH), 4.83 (q,

J=5.9 Hz, 1H; Pro CH), 5.15 (m, 2H; Pro NCH₂CH₂CH₂), 5.29 (s, 2H;

OCH₂C₆H₅), 7.07–7.40 (m, 12H; COOCH₂C₆H₅, NHCHACHTUNTRGNEUNG(CH₂C₆H₅)CO,

NH, NH), 7.73 (t, J=5.8 Hz, 1H; NH), 8.29 ppm (bt, J=5.7 Hz, 1H;

NH); ¹³C NMR: d=14.3, 22.9, 24.7, 27.1, 27.3, 27.8, 29.1, 29.5, 29.6, 29.6,

29.7, 29.8, 29.9, 32.1, 37.8, 43.2, 46.6, 47.0, 53.4, 53.6, 60.1, 67.3, 69.9,

127.1, 128.5, 128.6, 128.6, 128.8, 129.5, 135.5, 136.4, 142.4, 168.2, 168.7,

169.4, 170.0, 171.3, 171.7 ppm.

CH₂CH₃), 1.15–1.45 (m, 17H; OCH₂CH

2.20 (m, 6H; OCH₂CH₂A(CH₂)₄CH₃and Pro NCH₂CH₂CH₂), 2.80–3.50

(m, 4H; Trp CH₂and Pro NCH₂CH₂CH₂), 3.80–4.20 (m, 7H; Gly CH₂,

₂A

₂ACHTUNTGERNNUG(CH₂)₄CH₃and CACHTUNGTERN(NUGN CH₃)₃), 1.55–

CTHUNGTRENNUNG

OCH₂CH CHUTGNTREN(NNUG CH₂)₄CH₃and Pro CH), 4.80 (brq, J=6.7 Hz, 1H; Trp CH),

G

6.70–9.20 ppm (m, 9H; CONH, indole CH and CHO); ¹³C NMR: d=

14.3, 22.8, 24.8, 26.0, 28.3, 28.7, 29.1, 29.7, 31.9, 41.5, 43.5, 47.4, 53.7, 61.1,

65.8, 81.3, 116.7, 118.6, 124.9, 125.8, 131.6, 134.5, 156.0, 160.0, 169.3,

169.9, 171.8, 173.0 ppm.

(0.46 g,

0.40 mmol) in hot EtOH (40 mL) and 10% Pd/C (0.14 g) were placed in

a Parr shaker. The suspension was shaken (60 psi H₂, 3 h), the mixture

was filtered (celite pad) and the solvent was evaporated to give a white

solid (0.42 g, 98%). ¹H NMR: d=0.88 (t, J=6.9 Hz, 6H; CH₂CH₃), 1.25

AHCTUNGTRENNUNG

(brs, 60H; NCH₂CH

1.82–1.92 (m, 4H; Pro NCH₂CH₂CH₂), 3.04–3.49 (m, 6H; NCH₂CH₂-

(CH₂)₁₅CH₃, Phe NHCH(CH₂C₆H₅)CO), 3.71–4.12 (m, 8H; Gly CH₂,

COCH₂O), 4.28 (s, 2H; COCH₂O), 4.47 (m, 2H; Phe NHCH), 4.82 (q,

J=5.9 Hz, 1H; Pro CH), 5.15 (m, 2H; Pro NCH₂CH₂CH₂), 5.29 (s, 2H;

₂ACHTUNGTRENNUNG(CH₂)₁₅CH₃), 1.50 (brs, 4H; NCH₂CH₂AHCTUNTGRENN(GUN CH₂)₁₅CH₃),

AHCTUNGTRENNUNG

(C₁₈H₃₇)₂NCOCH₂OCH₂CO-GGG-OH^[24](0.53 g, 0.65 mmol), EDCI

(0.14 g, 0.72 mmol) and HOBt (0.10 g, 0.72 mmol) were suspended in

CH₂Cl₂(30 mL), cooled at 08C, Et₃N (0.27 mL) was added, and the mix-

ture stirred at RT for 90 h. The solvent was evaporated, the residue was

crystallized from MeOH, and further purified by chromatography (silica

gel, CHCl₃/MeOH=95:5–90:10). The NMR spectra indicated that the

G

ACHTUNGTRENNUNG

OCH₂C₆H₅), 7.17–7.25 (m, 5H; NHCHACHTNUGTREN(UNG CH₂C₆H₅)CO), 7.70 (brs, 1H;

NH), 7.84 (brs, 1H; NH), 8.24 (brs, 1H; NH), 8.33 ppm (brs, 1H; NH);

¹³C NMR: d=14.3, 22.9, 27.1, 27.2, 27.8, 29.0, 29.6, 29.6, 29.8, 29.9, 32.1,

43.2, 46.7, 47.2, 53.6, 126.9, 128.5, 129.6, 168.8, 170.2, 170.4, 170.6, 170.9,

171.3 ppm.

product was

CHO)GG-OC₇H₁₅

a

mixture of (C₁₈H₃₇)₂NCOCH₂OCH₂CO-GGGPW

ACHTUNGTRENNUNG(N-

and (C₁₈H₃₇)₂NCOCH₂OCH₂CO-GGGPWGG-

OC₇H₁₅(0.36 g, 41%). A portion (0.15 g) was dissolved in THF, and

NaOH (1.5 mL, 0.1m) was added dropwise. The mixture was stirred at

RT (40 min). Acetic acid was added (pH ca. 5) and the solvents evaporat-

ed. The residue was dissolved in CH₂Cl₂(15 mL) at 08C. EDCI (0.024 g,

0.12 mmol), DMAP (0.010 g, 0.08 mmol), and 1-heptanol (0.018 mL,

ACHTUNGTRENNUNG

0.38 mmol) was added under N₂to dry CH₂Cl₂(40 mL) and cooled to

08C. Diglycyl heptyl ester tosylate salt (TsOH·Gly-Gly-OC₇H₁₅) (0.15 g,

Chem. Eur. J. 2012, 00, 0 – 0

ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

&

11

&

ÞÞ

These are not the final page numbers!

G. W. Gokel et al.

0.12 mmol) were added and the mixture stirred at RT for 24 h. Evapora-

tion of the solvent followed by chromatography (silica gel, CHCl₃/

MeOH=93:7- 90:10) and crystallization from MeOH gave an off-white

solid (0.14 g, 95%, two steps). ¹H NMR: d=0.88 (t, J=6.6 Hz, 9H;

NaHCO₃(2ꢃ20 mL), brine (2ꢃ20 mL), dried over MgSO₄and the sol-

vent was evaporated. The residue was subjected to chromatography

(silica gel, EtOAc/hexane=40: 60) to give an oil (0.42 g, 83%).

¹H NMR: d=0.87 (t, J=6.3 Hz, 3H; CH₂CH₃), 1.20–1.35 (m, 8H;

CH₂CH₃), 1.15–1.40 (m, 68H; NCH₂CH

(CH₂)₄CH₃), 1.41–1.67 (m, 6H; NCH₂CH

(CH₂)₄CH₃), 1.72–2.10 (m, 4H; Pro NCH₂CH₂CH₂), 2.87–3.70 (m, 8H;

NCH₂CH₂A(CH₂)₁₅CH₃, Trp CH₂and Pro NCH₂CH₂CH₂), 3.86–4.38 (m,

17H; Gly CH₂, Pro CH, OCH₂CH₂A(CH₂)₄CH₃and OCH₂CO), 4.61 (q,

J=5.8 Hz, 1H; Trp CH), 6.64 (d, J=6.3 Hz, 1H; Trp CONH), 7.00–7.15

(m, 3H; indole CH and CONH), 7.18 (t, 1H; J=7.6 Hz, indole CH), 7.33

(brt, J=7.3 Hz, 1H; CONH), 7.41 (d, J=8.1 Hz, 1H; indole CH), 7.45–

7.57 (m, 2H; indole CH and CONH), 8.05 (brs, 1H; CONH), 8.46 (t, J=

5.7 Hz, 1H; CONH), 9.86 ppm (s, 1H; indole NH). ¹³C NMR: d=14.2,

14.3, 22.8, 22.9, 24.8, 25.9, 26.0, 27.1, 27.3, 27.8, 28.7, 28.9, 29.0, 29.1, 29.4,

29.5, 29.6, 29.7, 29.8, 29.9, 31.9, 32.1, 41.5, 41.9, 43.1, 43.3, 46.6, 46.7, 47.1,

54.7, 61.6, 65.8, 69.8, 71.9, 108.2, 112.6, 117.7, 119.6, 122.1, 124.8, 128.0,

136.4, 168.9, 169.0, 169.8, 170.1, 170.4, 170.7, 171. 172.4 ppm; elemental

analysis calcd (%) for C₇₃H₁₂₅N₉O₁₁: C 67.20, H 9.66, N 9.66; found: C

67.12, H 9.53, N 9.59.

₂A

OCH₂CH

(CH₃)₃), 1.63 (quint, J=6.6 Hz, 2H; OCH₂CH

5.1 Hz, 2H; Gly CH₂), 4.13 (t, J=6.8 Hz, 2H; OCH₂CH CTHUNGTRENNNUG

(CH₂)₄CH₃), 1.37 (d, J=7.2 Hz, 3H; Ala CH₃), 1.44 (s, 9H; C-

₂A(CH₂)₄CH₃), 4.02 (d, J=

₂A(CH₂)₄CH₃),

G

CHTUNGTRENNUNG

E

CHTUNGTRENNUNG

ACHTUNGTRENNUNG

CHTUNGTRENNUNG

4.23 (brquint, J=6.6 Hz, 1H; Ala CH), 5.11 (d, J=6.6 Hz, 1H; Ala NH),

6.79 ppm (brs, 1H; Gly NH); ¹³C NMR: d=14.2, 18.5, 22.8, 26.0, 28.5,

28.7, 29.0, 31.9, 41.5, 65.9, 80.5, 100.2, 170.0, 173.0 ppm; IR (KBr): n˜ =

3320, 3088, 2958, 2932, 2859, 1753, 1715, 1668, 1531, 1455, 1392, 1367,

CTHUNGTRENNUNG

1291, 1250, 1172, 1068, 1048, 1028 cm^ꢀ1

.

Boc-GAG-OC₇H₁₅: Boc-AG-OC₇H₁₅was treated with 4n HCl in dioxane

for 1 h. HCl·AG-OC₇H₁₅(0.49 g, 1.75 mmol), Boc-Gly-OH (0.31 g,

1.75 mmol), EDCI (0.37 g, 1.92 mol) and HOBt (0.26 g, 1.92 mmol) were

dissolved in CH₂Cl₂(30 mL) and Et₃N (0.73 mL) was then added. The

mixture was stirred at 08C for 0.5 h and at RT for 12 h. The solvent was

removed in vacuo. The residue was dissolved in CH₂Cl₂(40 mL), washed

with 5% citric acid (2ꢃ20 mL), H₂O (2ꢃ20 mL), 5% NaHCO₃(2ꢃ

20 mL), brine (2ꢃ20 mL), dried over MgSO₄and the solvent was evapo-

rated. Chromatography (silica gel, CHCl₃/CH₃OH=97:3) gave an oil

(0.50 g, 71%). ¹H NMR: d=0.88 (t, J=6.3 Hz, 3H; CH₂CH₃), 1.30

Compound 5

Boc-(d)-Trp-Gly-Gly-OC₇H₁₅

: Boc-(d)-Tryptophan (0.25 g, 0.82 mmol)

(pseudo-s, 8H; OCH₂CH

1.45 (s, 9H; C(CH₃)₃), 1.64 (quint, J=6.2 Hz, 2H; OCH₂CH

3.83 (pseudo-s, 2H; Gly CH₂), 4.01 (t, J=5.0 Hz, 2H; Gly CH₂), 4.13 (t,

J=6.8 Hz, 2H; OCH₂CH₂A(CH₂)₄CH₃), 4.60 (quint, J=7.0 Hz, 1H; Ala

(CH₂)₄CH₃), 1.41 (d, J=6.9 Hz, 3H; Ala CH₃),

was dissolved in dry CH₂Cl₂(30 mL), cooled to 08C and diglycyl heptyl

ester tosylate salt^[29](TsOH·Gly-Gly-OC₇H₁₅, 0.33 g, 0.82 mmol), EDCI

(0.17 g, 0.90 mmol, HOBt (0.12 g, 0.90 mmol, and NEt₃(0.25 g,

2.46 mmol) were added. The reaction was stirred at 08C for 30 min and

then at RT for 24 h. The solvent was evaporated, the resulting yellow oil

was subjected to chromatography (silica gel, 98:2 CH₂Cl₂/MeOH), and

the residue was crystallized from MeOH to afford an off-white solid

(0.25 g, 59%). ¹H NMR: d=0.88 (t, J=6.8 Hz, 3H; OCH₂CH₂-

N

CHTUNGTRENNUNG

CTHUNGTRENNUNG

CH), 5.33 (1H; brs, Gly NH), 6.88 (1H; d, J=7.5 Hz, Ala NH),

6.99 ppm (1H; brs, Gly NH); ¹³C NMR: d=14.2, 18.3, 22.8, 26.0, 28.5,

28.7, 29.0, 31.9, 41.5, 44.5, 48.9, 65.9, 80.6, 169.8, 170.0, 172.5 ppm; IR

(KBr): n˜ =3310, 3076, 2958, 2932, 2859, 1743, 1717, 1658, 1529, 1455,

1392, 1367, 1283, 1249, 1174, 1052, 1021 cm^ꢀ1

.

A

₂CATHUNGTERN(UNNG CH₂)₄CH₃), 1.40 (s, 9H; (CH₃)₃),

CTHUNGTRENNUNG

Boc-PGAG-OC₇H₁₅: Boc-GAG-OC₇H₁₅was stirred with 4n HCl in diox-

ane for 1 h. HCl·GAG-OC₇H₁₅(0.40 g, 1.18 mmol), Boc-Pro-OH (0.25 g,

1.18 mmol), EDCI (0.25 g, 1.30 mmol) and HOBt (0.18 g, 1.30 mmol)

were dissolved in CH₂Cl₂(35 mL) and Et₃N (0.50 mL) was added. The

mixture was stirred at 08C for 0.5 h and at RT for 48 h. Evaporation of

the solvent and chromatography (silica gel, CHCl₃/CH₃OH=97:3) afford-

ACHTUNGTRENNUNG

NH), 7.06–7.18 (m, 3H; indole aromatic CH), 7.32–7.35 (m, 1H; indole

aromatic CHNH), 7.58–7.60 (m, 1H; indole aromatic CHNH), 8.49 ppm

(brs, 1H; NH). ¹³C NMR: d=14.3, 22.8, 25.9, 28.5, 28.7, 29.1, 31.9, 41.3,

43.2, 65.9, 77.4, 110.3, 111.6, 118.9, 119.9, 122.5, 123.6, 127.5, 136.4, 169.8,

169.9, 172.8 ppm.

1

ed a light yellow solid (0.51 g, 86%). M.p. 159–1618C; H NMR: d=0.89

(t, J=6.6 Hz, 3H; CH₂CH₃), 1.20–1.38 (m, 8H; OCH₂CH

1.40–1.50 (m, 12H; Ala CH₃and C(CH₃)₃), 1.82–2.22 (m, 4H; Pro

NCH₂CH₂CH₂), 3.40–3.52 (m, 2H; Pro NCH₂CH₂CH₂), 3.93–4.02 (m,

4H; two Gly CH₂), 4.12 (t, J=6.6 Hz, 2H; OCH₂CH₂A(CH₂)₄CH₃), 4.21

₂ACHTUNGRTNE(NUNG CH₂)₄CH₃),

ACHTUNGTRENNUNG(C₁₈H₃₇)₂NCOCH₂OCH₂CO-Gly-Gly-Gly-Pro-(d)-Trp-Gly-Gly-OC₇H₁₅

AHCTUNGTRENNUNG

(5): Boc-(d)-WGG-OC₇H₁₅(0.30 g, 0.61 mmol) was stirred with 4n HCl/

dioxane for 2 h. HCl·(d)-WGG-OC₇H₁₅, (C₁₈H₃₇)₂NCOCH₂OCH₂CO-

GGGP-OH (0.53 g, 0.61 mmol, see above for synthesis), EDCI (0.13 g,

0.68 mmol) and HOBt (0.09 g, 0.68 mmol) were suspended in CH₂Cl₂

(30 mL). The mixture was cooled to 08C, Et₃N (0.19 g) was added, and

stirring was continued at RT for 3 d. Evaporation of the solvent and

chromatography (silica gel, CHCl₃/MeOH=93:7) followed by crystalliza-

tion from MeOH gave an off-white solid (0.35 g, 48%). ¹H NMR: d=

0.87 (t, J=6.8 Hz, 9H; CH₂CH₃), 1.25–1.61 (m, 74H; NCH₂CH₂-

CTHUNGTRENNUNG

(dd, J=7.8, 5.4 Hz, 1H; Pro CH), 4.54 (quint, J=7.4 Hz, 1H; Ala CH),

7.00 (brs, 2H; two Gly NH), 7.34 ppm (brd, J=7.4 Hz, 1H; Ala NH);

¹³C NMR: d=14.3, 17.6, 22.8, 25.3, 26.0, 28.6, 28.7, 29.1, 29.9, 31.9, 41.5,

43.2, 47.6, 49.4, 61.2, 65.7, 81.2, 173.7 ppm; IR (KBr): n˜ =3307, 2957,

2930, 1752, 1668, 1535, 1454, 1407, 1367, 1165, 1133, 1019 cm^ꢀ1

(C₁₈H₃₇)₂NCOCH₂OCH₂CO-GGGPGAG-OC₇H₁₅(6):

OC₇H₁₅was deprotected (4n HCl, dioxane,

.

A

Boc-PGAG-

1 h).

A

OCH₂CH

(CH₂)₄CH₃,

NCH₂CH

(CH₂)₁₅CH₃

and

(C₁₈H₃₇)₂NCOCH₂OCH₂CO-GGG-OH (0.44 g, 0.54 mmol), HCl·PGAG-

OC₇H₁₅(0.25 g, 0.54 mmol), EDCI (0.11 g, 0.60 mmol), and HOBt

(0.08 g, 0.60 mmol) were suspended in CH₂Cl₂(35 mL) and Et₃N

(0.23 mL) was added. The mixture was stirred at 08C for 0.5 h and at RT

for 48 h. Evaporation of the solvent and chromatography (silica gel,

CHCl₃/CH₃OH/HOAc=90: 10: 0.1) gave a white solid (0.19 g, 29%).

M.p. 128–1308C; ¹H NMR: d=0.88 (t, J=6.4 Hz, 9H; CH₂CH₃), 1.20–

CHTUNGTRENNUNG

CTHUNGTRENNUNG

N

CHTUNGTRENNUNG

3.67–4.26, (m, 14H; Gly CH_2,OCH₂CO), 4.59 (q, J=5.5 Hz, 1H; Trp

CH), 7.04–7.44 (m, 6H; indole CH, NH), 7.57 (d, J=7.8 Hz, 1H; Trp

CONH), 7.79 (t, J=5.8 Hz, 1H; NH), 7.87 (t, J=5.8 Hz, 1H; NH), 8.30

(t, J=5.8 Hz, 1H; NH), 9.18 ppm (1H; s, indole CH);¹³C NMR: d=14.2,

14.3, 22.8, 22.9, 25.4, 26.0, 26.5, 27.1, 27.3, 27.8, 29.1, 29.6, 31.9, 32.1, 41.4,

41.8, 43.2, 46., 47.1, 51.0, 55.1, 61.2, 65.9, 69.6, 71.6, 109.7, 111.8, 118.6,

119.7, 122.2, 123.9, 127.8, 136.5, 168.6, 170.1, 170.3, 170.4, 171.0, 172.2,

173.4 ppm.

1.40 (m, 71H; CH

CH₃), 1.42–1.67 (m, 6H; CH

(CH₂)₄CH₃), 1.92–2.21 (m, 4H; Pro NCH₂CH₂CH₂), 3.09 (t, 2H; J=

7.2 Hz, CH₃A(CH₂)₁₅CH₂CH₂N), 3.28 (t, J=7.5 Hz, 2H; CH₃-

(CH₂)₁₅CH₂CH₂N), 3.50–3.80 (m, 3H; Pro NCH₂CH₂CH₂and Gly CH₂),

3.85–4.15 (m, 13H; Gly CH₂, COCH₂O and OCH₂CH₂A(CH₂)₄CH₃), 4.29

(CH₂)₁₅CH₂CH₂N, OCH₂CH

₂ACHTUNGTNERN(UNG CH₂)₄CH₃and Ala

CTHUNGTRENNUNG

AHCTUNGTRENNUNG

CTHUNGTRENNUNG

AHCTUNGTRENNUNG

Compound 6

CTHUNGTRENNUNG

[29]

(s, 2H; COCH₂O), 4.40–4.51 (m, 2H; Pro CH and Ala CH). 7.38–7.50

(m, 2H; two CONH), 7.54 (brt, J=5.8 Hz, 1H; CONH), 7.90–8.02 (m,

2H; two CONH), 8.26 ppm (brt, J=5.8 Hz, 1H; CONH); ¹³C NMR: d=

14.3, 17.7, 20.8, 22.8, 22.9, 25.3, 26.0, 27.1, 27.3, 27.8, 28.7, 29.1, 29.5, 29.6,

29.8, 29.9, 31.9, 32.1, 41.5, 43.2, 46.6, 47.2, 49.2, 51.2, 61.3, 65.8, 69.8, 71.8,

76.2, 100.2, 164.9, 168.2, 168.8, 169.7, 170.2, 170.3, 171.5, 172.8,

Boc-AG-OC₇H₁₅

:

TsOH·Gly-OC₇H₁₅

(0.51 g, 1.49 mmol), Boc-l-Ala

(0.28 g, 1.48 mmol), EDCI (0.31 g, 1.62 mmol) and HOBt (0.22 g,

1.63 mmol) were dissolved in CH₂Cl₂(40 mL) and Et₃N (0.61 mL) was

added. The mixture was stirred at 08C for 0.5 h and then at RT for 48 h.

The solvent was evaporated and the residue was dissolved in CH₂Cl₂

(40 mL), washed with 5% citric acid (2ꢃ20 mL), H₂O (2ꢃ20 mL), 5%

&

12

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Chem. Eur. J. 0000, 00, 0 – 0

ÝÝ

These are not the final page numbers!

Anion Transport

FULL PAPER

173.3 ppm; IR (KBr): n˜ =3307, 3081, 2923, 2853, 1749, 1657, 1549, 1466,

43.6, 46.5, 47.1, 53.0, 61.1, 65.8, 69.7, 71.7, 116.1, 118.5, 119.2, 124.5, 124.6,

125.3, 131.6, 134.2, 160.6, 168.5, 168.7, 170.1, 171.5, 171. 173.0 ppm; ele-

mental analysis calcd (%) for C₇₄H₁₂₅N₉O₁₂: C 66.68, H 9.45, N 9.46;

found: C 66.82, H 9.30, N 9.20.

1377, 1243, 1201, 1130, 1026, 721, 666 cm^ꢀ1

.

Compound 7: (C₁₈H₃₇)₂NCOCH₂OCH₂CO-Gly-Gly-Gly-Pro-Gly-Glu-

Gly-OC₇H₁₅(7) was prepared as previously reported.^[29]

Fraction 2 was reduced to minimum volume and crystallized from MeOH

to give an off-white solid (80 mg, 13%). This fraction was identified as

(C₁₈H₃₇)₂NCOCH₂OCH₂CO -GGGPGWG-OC₇H₁₅. ¹H NMR (300 MHz;

all peaks were broad): d=0.88 (t, J=7.0 Hz, 9H; CH₂CH₃), 1.10–1.70

Compound 8

Boc-WACHTUNGTRENNUNG(N-CHO)G-OC₇H₁₅: Boc-WCAHTUNGRTENN(UGN N-CHO)-OH (0.60 g, 1.80 mmol),

glycine n-heptyl ester tosylate^[29](0.62 g, 1.80 mmol), EDCI (0.38 g,

1.99 mmol) and HOBt (0.27 g, 1.99 mmol) were dissolved in CH₂Cl₂

(35 mL), and cooled to 08C. Et₃N (0.75 mL) was added and the mixture

was stirred at RT for 72 h. Evaporation of the solvent and chromatogra-

phy (silica gel, hexane/EtOAc=2:1) afforded an oil (0.62 g, 70%).

¹H NMR: d=0.88 (t, J=6.7 Hz, 3H; CH₂CH₃), 1.20–1.36 (m, 8H;

(m, 74H; NCH

Pro NCH₂CH₂CH₂), 2.93–4.50 (m, 25H; NCH₂CH

Pro NCH₂CH₂CH₂, Gly CH₂, OCH₂CH₂A(CH₂)₄CH₃, Pro CH and

₂ACHUTGTERNNGU(CH₂)₁₆CH₃and OCH₂ACHTUNTGRNE(NUNG CH₂)₅CH₃), 1.80–2.20 (m, 4H;

CTHUNGTRENNUNG

OCH₂CO), 4.78 (brs, 1H; Trp CH), 6.95–8.30 (m, 11H; indole CH and

CONH), 9.53 ppm (s, 1H; indole NH); ¹³C NMR: d=14.2, 14.3, 22.8,

22.9, 24.9, 26.0, 27.1, 27.3, 27.8, 28.7, 29.1, 29.5, 29.6, 29.8, 29.9, 31.9, 32.1,

41.5, 42.2, 42.8, 46.6, 46.8, 47.2, 54.1, 61.1, 65.6, 69.7, 71.6, 110.6, 111.9,

118.7, 118.9, 121.6, 124.4, 127.6, 136.6, 168.5, 168.6, 170.0, 170.2, 170.4,

170.5, 171.2, 172. 173.1 ppm; elemental analysis calcd (%) for

C₇₃H₁₂₅N₉O₁₁: C 67.20, H 9.66, N 9.66; found: C 67.42, H 9.41, N 9.47.

OCH₂CH CHTUNGTRENNUNG(CH₂)₄CH₃), 1.39 (s, 9H; CAHCTNUGTRENGN(UN CH₃)₃), 1.50–1.70 (m, 2H;

₂A

₂A(CH₂)₄CH₃), 3.10–3.33 (m, 2H; Trp CH₂), 3.96 (d, J=5.3 Hz,

₂A(CH₂)₄CH₃), 4.57 (brs,

OCH₂CH CHTUNGTRENNUNG

2H; Gly CH₂), 4.09 (t, J=6.8 Hz, 2H; OCH₂CH HCTUNGTRNENNUG

1H; Trp CH), 5.29 (d, J=8.2 Hz, 1H; NH), 6.65–9.10 ppm (m, 7H;

CONH, indole CH and CHO); ¹³C NMR: d=14.2, 22.7, 25.9, 28.4, 28.6,

29.0, 31.8, 41.4, 54.3, 65.9, 80.6, 116.4, 118.6, 119.2, 124.1, 124.8, 125.6,

131.4, 134.5, 155.6, 159.6, 169.7, 171.6 ppm.

Compound 9: (C₁₈H₃₇)₂NCOCH₂OCH₂CO-Gly-Gly-Gly-Pro-Gly-Gly-

Boc-GW

N

:

Boc-W

E

(0.60 g,

GluACTHUNGETRNNUG

(OBz)-OC₇H₁₅(9) was prepared as reported.^[29]

1.23 mmol) was deprotected (4n HCl/dioxane, 2 h). HCl·WAHCTUNGTRENNUNG

Compound 10: (C₁₈H₃₇)₂NCOCH₂OCH₂CO-Gly-Gly-Gly-Pro-Gly-Gly-

Glu(OCH₂pyrene)-OC₇H₁₅(10) was prepared as previously reported.^[29]

OC₇H₁₅, Boc-Gly-OH (0.22 g, 1.23 mmol), EDCI (0.26 g, 1.35 mmol) and

HOBt (0.18 g, 1.35 mmol) were dissolved in CH₂Cl₂(35 mL), cooled at

08C and Et₃N (0.51 mL) was added. The mixture was stirred at RT for

72 h. Evaporation of the solvent and chromatography (silica gel, CHCl₃/

Compound 11: (C₁₈H₃₇)₂NCOCH₂OCH₂CO-Gly-Gly-Gly-Pro-Gly-Gly-

LysACHTNUTGRNEUNG

(NBD)-OC₇H₁₅(11) was prepared as previously reported.^[29]

1

Compounds 12 and 13

MeOH=98:2) gave an oil (0.42 g, 63%). H NMR: d=0.88 (t, J=6.5 Hz,

3H; CH₂CH₃), 1.20–1.34 (m, 8H; OCH₂CH

(CH₃)₃), 1.50–1.71 (m, 2H; OCH₂CH₂A(CH₂)₄CH₃), 3.13–3.31 (m, 2H; Trp

CH₂), 3.74–4.00 (m, 4H; Gly CH₂), 4.07 (t, J=6.6 Hz, 2H; OCH₂CH₂-

₂ACHTUNGTRENNUN(G CH₂)₄CH₃), 1.37 (s, 9H; C-

N-Formyl l-tryptophan n-heptyl ester tosylate: A mixture of l-TrpACHTUNGTRENNUNG(N-

G

CHTUNGTRENNUNG

CHO)-OH (0.60 g, 2.75 mmol), TsOH·H₂O (0.58 g, 3.02 mmol), 1-hepta-

nol (3.1 mL, 22.0 mmol), and toluene (40 mL) was heated to reflux and

water removed (Dean–Stark) during 10 h. The mixture was cooled to RT,

Et₂O (200 mL) was added, and the mixture was further cooled to 08C

(16 h). Filtration gave a white solid (0.78 g, 58%). ¹H NMR: d=0.88 (t,

ACHTUNGTRENNUNG(CH₂)₄CH₃), 4.91 (d, J=7.0 Hz, 1H; Trp CH), 5.36 (brs, 1H; NH), 7.00–

9.10 ppm (m, 8H; CONH, indole CH and CHO); ¹³C NMR: d=14.4,

22.9, 26.1, 27.9, 28.6, 28.9, 29.2, 32.0, 41.6, 44.8, 53.1, 66.1, 80.8, 116.6,

118.3, 119.2, 124.8, 124.9, 125.7, 131.6, 134.6, 156.7, 160.2, 170.0, 170.4,

171.6 ppm.

J=6.9 Hz, 3H; CH₂CH₃), 1.00–1.45 (m, 10H; OCH

3H; CH₃C₆H₄SO₃), 3.30–3.40 (m, 2H; Trp CH₂), 3.49 (s, 3H; indole

NCH₃), 3.73–3.98 (m, 3H; OCH₂CH₂A(CH₂)₄CH₃and Trp CH), 6.96–7.30

₂ACHTUNGTRNE(UNNG CH₂)₅CH₃), 2.31 (s,

Boc-PGW

G

:

Boc-GW

U

CHTUNGTRENNUNG

0.64 mmol) was deprotected (4N HCl/dioxane, 2 h). HCl·GWAHCTUNGTRENNUNG

(m, 6H; indole CH and H_Ar), 7.42 (d, J=7.8 Hz, 1H; indole CH), 7.60

(d, J=7.8 Hz, 2H; H_Ar), 8.13 ppm (brs, 3H; Trp NH₃); ¹³C NMR: d=

14.3, 21.5, 22.8, 25.7, 26.2, 28.3, 29.1, 31.9, 32.6, 53.6, 66.7, 105.3, 109.6,

118.6, 119.2, 121.7, 126.2, 127.7, 129.0, 130.0, 137.2, 140.5, 169.2 ppm.

CHO)G-OC₇H₁₅, Boc-Pro-OH (0.14 g, 0.64 mmol), EDCI (0.14 g,

0.71 mmol), and HOBt (0.095 g, 0.71 mmol) were dissolved in CH₂Cl₂

(25 mL), cooled at 08C and Et₃N (0.27 mL) was added. The mixture was

stirred at RT for 64 h. Evaporation of the solvent and chromatography

Boc-GW

N

:

TsOH·W

G

(0.65 g,

1

(silica gel, CHCl₃/MeOH=98:2) gave an oil (0.31 g, 75%). H NMR: d=

1.33 mmol), Boc-Gly-OH (0.23 g, 1.33 mmol), EDCI (0.28 g, 1.46 mmol),

and HOBt (0.20 g, 1.46 mmol) were dissolved in CH₂Cl₂(35 mL), cooled

at 08C and Et₃N (0.56 mL) was added. The mixture was stirred at RT for

24 h, the solvent was evaporated, and the residue was subjected to chro-

matography (silica gel, hexane/EtOAc=2:1) to afford an oil (0.39 g,

62%). ¹H NMR: d=0.88 (t, J=6.2 Hz, 3H; CH₂CH₃), 1.27 (pseudo-s,

0.88 (t, J=6.8 Hz, 3H; CH₂CH₃), 1.15–1.45 (m, 17H; OCH₂CH₂-

A

ACHTUNGTRENNUNG(CH₃)₃), 1.50–2.18 (m, 6H; OCH₂CH₂HCAUTNGTREN(NUGN CH₂)₄CH₃and

CTHUNGTRENNUNG

(brs, 1H; Trp CH), 6.80–9.20 ppm (m, 9H; CONH, indole CH and

CHO); ¹³C NMR: d=14.2, 22.8, 24.8, 26.0, 27.2, 28.5, 28.7, 29.1, 29.6,

31.9, 41.5, 43.7, 47.6, 53.0, 60.8, 65.8, 81.0, 116.3, 119.1, 124.7, 125.5, 131.5,

134.5, 155.8, 160.1, 169.9, 171.4, 173.9 ppm.

8H; OCH₂CH

N

ACHTUNGTREN(NNUG CH₃)₃), 1.57 (quint, J=6.3 Hz,

2H; OCH₂CH

₂A

J=5.3 Hz, 2H; Gly CH₂), 3.70–3.85 (m, 5H; Gly CH₂and indole NCH₃),

3.95–4.15 (m, 2H; OCH₂CH₂A(CH₂)₄CH₃), 4.90 (q, J=6.1 Hz, 1H; Trp

ACHTUNGTRENNUNG

CTHUNGTRENNUNG

ACHTUNGTRENNUNG

CH), 5.07 (brs, 1H; NH), 6.46–7.55 ppm (m, 6H; CONH and indole

CH); ¹³C NMR: d=14.2, 22.8, 25.9, 27.8, 28.4, 28.6, 29.0, 31.9, 32.9, 44.4,

53.3, 65.9, 80.3, 108.4, 109.5, 118.7, 119.4, 122.0, 127.7, 128.4, 137.1, 156.0,

169.1, 171.9 ppm.

AHCTUNGTRENNUNG

(C₁₈H₃₇)₂NCOCH₂OCH₂CO-GGG-OH (0.38 g, 0.47 mmol), EDCI

(0.098 g, 0.51 mmol), and HOBt (0.069 g, 0.51 mmol) were suspended in

CH₂Cl₂(25 mL) and Et₃N (0.20 mL) was added (08C). The mixture was

stirred at RT for 60 h. The solvent was evaporated and the residue was

crystallized from MeOH. Chromatography (silica gel, CHCl₃:MeOH=

98:2–95: 5–93:7) afforded two major fractions. The first fraction was re-

duced in volume and crystallized from MeOH to afford a white solid

Boc-GGW (0.38 g,

A

:

Boc-GW

N

0.80 mmol) was deprotected (4n HCl/dioxane, 2 h). HCl·GWACHTUNGTRENNUNG(N-CH₃)-

OC₇H₁₅, Boc-Gly-OH (0.14 g, 0.80 mmol), EDCI (0.17 g, 0.88 mmol), and

HOBt (0.12 g, 0.88 mmol) were suspended in CH₂Cl₂(30 mL), the mix-

ture was cooled to 08C, and Et₃N (0.34 mL) was added. The mixture was

stirred at RT for 40 h. Evaporation of the solvent and chromatography

(silica gel, CHCl₃/MeOH=98:2) afforded a viscous solid (0.24 g, 56%).

¹H NMR: d=0.88 (t, J=6.6 Hz, 3H; CH₂CH₃), 1.25 (pseudo-s, 8H;

(130 mg, 21%) identified as (C₁₈H₃₇)₂NCOCH₂OCH₂CO-GGGPGW

CHO)G-OC₇H₁₅. ¹H NMR (300MHz; all peaks were broad): d=0.88 (t,

J=6.9 Hz, 9H; CH₂CH₃), 1.00–1.71 (m, 74H; NCH₂A(CH₂)₁₆CH₃and

OCH₂A(CH₂)₅CH₃), 1.75–2.28 (m, 4H; Pro NCH₂CH₂CH₂), 2.93–4.50 (m,

25H; NCH₂CH₂A(CH₂)₁₅CH₃, Trp CH₂, Pro NCH₂CH₂CH₂, Gly CH₂,

OCH₂CH₂A(CH₂)₄CH₃, Pro CH and OCH₂CO), 4.80 (brs, 1H; Trp CH),

ACHTUNGERTN(NUNG N-

CTHUNGTRENNUNG

CHTUNGTRENNUNG

OCH₂CH CHUTTGNERNN(UGN CH₂)₄CH₃), 1.44 (s, 9H; CAHCTNUGTRENGN(UN CH₃)₃), 1.50–1.63 (m, 2H;

₂A

CHTUNGTRENNUNG

OCH₂CH₂AHCUTGNTRN(ENUNG CH₂)₄CH₃), 3.20–3.35 (m, 2H; Trp CH₂), 3.64–3.78 (m, 5H;

CHTUNGTRENNUNG

Gly CH₂and indole NCH₃), 3.84–4.12 (m, 4H; Gly CH₂and OCH₂CH₂-

(CH₂)₄CH₃), 4.85 (q, J=6.2 Hz, 1H; Trp CH), 5.19 (brs, 1H; NH), 6.60–

7.55 (m, 7H; CONH and indole CH). ¹³C NMR: d=14.2, 22.7, 25.9, 27.6,

7.20–8.45 (m, 11H; indole CH and CONH), 9.11 ppm s, (1H; CHO);

¹³C NMR (CDCl₃): d=14.2, 14.3, 22.8, 22.9, 25.2, 26.0, 26.8, 27.1, 27.2,

27.8, 28.7, 29.1, 29.5, 29.6, 29.7, 29.8, 29.9, 31.9, 32.1, 41.5, 41.9, 42.9, 43.3,

AHCTUNGTRENNUNG

Chem. Eur. J. 2012, 00, 0 – 0

ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

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G. W. Gokel et al.

28.5, 28.6, 29.0, 31.8, 32.9, 43.0, 44.3, 53.4, 65.9, 80.5, 108.4, 109.6, 118.7,

119.3, 122.0, 127.9, 128.3, 137.1, 156.2, 168.5, 170.1, 171.9 ppm.

1028 cm^ꢀ1; elemental analysis calcd (%) for C₇₃H₁₂₅N₉O₁₁: C 67.20, H

9.66, N 9.66; found: C 67.44, H 9.53, N 9.41.

Compound 14

Boc-PGGW

(N-CHO)-OC₇H₁₅

:

Boc-GGWACHUTNGTRENNUNG

0.41 mmol) was deprotected (4n HCl/dioxane, 2 h). HCl·GGWAHCTUNGTRENNUNG

N-Methyl l-tryptophan n-heptyl ester tosylate: A mixture of l-TrpACHTUNGTRENNUNG(N-

OC₇H₁₅, Boc-Pro-OH (0.089 g, 0.41 mmol), EDCI (0.087 g, 0.46 mmol),

and HOBt (0.062 g, 0.46 mmol) were suspended in CH₂Cl₂(25 mL) and

Et₃N (0.17 mL) was added. The mixture was stirred at RT for 24 h. Evap-

oration of the solvent and chromatography (silica gel, CHCl₃/MeOH=

98:2- 95:5) gave a viscous semi-solid (0.26 g, 98%). ¹H NMR: d=0.89 (t,

CH₃)-OH (0.60 g, 2.75 mmol), TsOH·H₂O (0.58 g, 3.02 mmol), 1-heptanol

(3.1 mL, 22.0 mmol) and toluene (40 mL) was heated to reflux (10 h,

Dean–Stark trap). The mixture was cooled to RT, Et₂O (200 mL) was

added and the mixture was maintained at 08C for 16 h. The solid product

1

was collected by filtration (0.78 g, 58%). H NMR: d=0.88 (t, J=6.9 Hz,

J=6.8 Hz, 3H; CH₂CH₃), 1.26 (pseudo-s, 8H; OCH₂CH

G

3H; CH₂CH₃), 1.00–1.45 (m, 10H; OCH

CH₃C₆H₄SO₃), 3.30–3.40 (m, 2H; Trp CH₂), 3.49 (s, 3H; indole NCH₃),

3.73–3.98 (m, 3H; OCH₂CH₂A(CH₂)₄CH₃and Trp CH), 6.96–7.30 (m, 6H;

indole CH and H_Ar), 7.42 (d, J=7.8 Hz, 1H; indole CH), 7.60 (d, J=

7.8 Hz, 2H; H_Ar), 8.13 ppm (brs, 3H; Trp NH₃); ¹³C NMR: d=14.3, 21.5,

22.8, 25.7, 26.2, 28.3, 29.1, 31.9, 32.6, 53.6, 66.7, 105.3, 109.6, 118.6, 119.2,

121.7, 126.2, 127.7, 129.0, 130.0, 137.2, 140.5, 169.2 ppm.

₂ACHTUNGTRENUN(GN CH₂)₅CH₃), 2.31 (s, 3H;

1.43 (s, 9H; C(CH₃)₃), 1.48–1.61 (m, 2H; OCH₂CH₂A(CH₂)₄CH₃), 1.82–

A

CHTUNGTRENNUNG

CTHUNGTRENNUNG

2.20 (m, 4H; Pro NCH₂CH₂CH₂), 3.28 (d, J=5.7 Hz, 2H; Trp CH₂),

3.34–3.55 (m, 2H; Pro NCH₂CH₂CH₂), 3.68–4.20 (m, 10H; Gly CH₂,

indole NCH₃, OCH₂CH₂ACHTNUGTRNEUNG(CH₂)₄CH₃and Pro CH), 4.83 (q, J=6.2 Hz,

1H; Trp CH), 6.73–7.65 ppm (m, 8H; CONH and indole CH); ¹³C NMR:

d=14.2, 22.8, 24.9, 25.9, 27.6, 28.6, 29.1, 29.6, 29.9, 31.8, 32.9, 43.3, 47.5,

53.3, 60.9, 65.8, 81.0, 108.6, 109.5, 118.8, 119.2, 121.8, 128.1, 128.4, 137.0,

155.9, 168.8, 169.9, 171.9, 173.5 ppm.

Boc-GWACTHUNTRGENN(UG N-CH₃)-OC₇H₁₅: TsOH·WACHUTTGNERN(NNGU N-CH₃)-OC₇H₁₅(0.65 g, 1.33 mmol),

Boc-Gly-OH (0.23 g, 1.33 mmol), EDCI (0.28 g, 1.46 mmol), and HOBt

(0.20 g, 1.46 mmol) were dissolved in CH₂Cl₂(35 mL), cooled at 08C, and

Et₃N (0.56 mL) was added. The mixture was stirred at RT for 24 h. Evap-

oration of the solvent and chromatography (silica gel, hexane/EtOAc=

2:1) afforded an oil (0.39 g, 62%). ¹H NMR: d=0.88 (t, J=6.2 Hz, 3H;

G

AHCTUNGTRENNUNG

(C₁₈H₃₇)₂DGA-GGG-OH (0.54 g, 0.67 mmol), EDCI (0.14 g, 0.74 mmol)

and HOBt (0.10 g, 0.74 mmol) were suspended in CH₂Cl₂(25 mL). To

this mixture cooled at 08C, was added Et₃N (0.28 mL). The mixture was

warmed to room temperature and stirred for 48 h. The solvent was

evaporated and the residue was crystallized from MeOH. The crude

product was further purified by column chromatography (silica gel,

CHCl₃/MeOH=95:5–90:10) and then recrystallized from MeOH to

afford a white solid (0.34 g, 38%). M.p. 172–1748C; ¹H NMR (CDCl₃):

d=0.88 (t, J=6.6 Hz, 9H; CH₂CH₃), 1.18–1.40 (m, 68H; NCH₂CH₂-

CH₂CH₃), 1.27 (pseudo-s, 8H; OCH₂CH

(CH₂)₄CH₃), 1.43 (s, 9H; C-

₂A(CH₂)₄CH₃), 3.32 (d, J=

G

5.3 Hz, 2H; Trp CH₂), 3.96 (d, J=5.3 Hz, 2H; Gly CH₂), 3.70–3.85 (m,

5H; Gly CH₂and indole NCH₃), 3.95–4.15 (m, 2H; OCH₂CH₂-

AHCTNUGTREN(GUNN CH₂)₄CH₃), 4.90 (q, J=6.1 Hz, 1H; Trp CH), 5.07 (brs, 1H; NH), 6.46–

7.55 ppm (m, 6H; CONH and indole CH); ¹³C NMR: d=14.2, 22.8, 25.9,

27.8, 28.4, 28.6, 29.0, 31.9, 32.9, 44.4, 53.3, 65.9, 80.3, 108.4, 109.5, 118.7,

119.4, 122.0, 127.7, 128.4, 137.1, 156.0, 169.1, 171.9 ppm.

Boc-GGW (0.38 g,

A

:

Boc-GW

N

A

(CH₂)₄CH₃), 1.40–1.64 (m, 6H; NCH₂CH₂-

₂A(CH₂)₄CH₃), 1.88–2.23 (m, 4H; Pro

₂A(CH₂)₁₅CH₃), 3.16–

0.80 mmol) was deprotected (4n HCl/dioxane, 2 h). HCl·GWACHTUNGTRENNUNG(N-CH₃)-

E

CHTUNGTRENNUNG

OC₇H₁₅, Boc-Gly-OH (0.14 g, 0.80 mmol), EDCI (0.17 g, 0.88 mmol), and

HOBt (0.12 g, 0.88 mmol) were suspended in CH₂Cl₂(30 mL), cooled to

08C, and Et₃N (0.34 mL) was added. The mixture was stirred at for 40 h.

Evaporation of the solvent and chromatography (silica gel, CHCl₃/

CTHUNGTRENNUNG

CHTUNGTRENNUNG

ACHTUNGTRENNUNG

1

MeOH=98:2) afforded a viscous semi-solid (0.24 g, 56%). H NMR: d=

9.40 ppm (m, 12H; CONH, indole CH and CHO); ¹³C NMR (CDCl₃):

d=14.2, 14.3, 22.8, 22.9, 25.4, 26.0, 27.1, 27.3, 27.8, 28.6, 29.1, 29.2, 29.5,

29.6, 29.8, 29.9, 30.0, 31.9, 32.1, 42.2, 43.2, 43.8, 46.6, 47.2, 52.3, 61.5, 66.2,

69.6, 71.6, 116.3, 118.1, 118.8, 124.5, 124.8, 125.3, 131.4, 134.4, 160.6,

168.8, 169.1, 170.2, 170.3, 170.4, 170.7, 171.4, 172.0, 173.8 ppm; elemental

analysis calcd (%) for C₇₄H₁₂₅N₉O₁₂: C 66.68, H 9.45, N 9.46; found: C

66.90, H 9.19, N 9.28.

0.88 (t, J=6.6 Hz, 3H; CH₂CH₃), 1.25 (pseudo-s, 8H; OCH₂CH₂-

A

CACHTGNUTERNNU(G CH₃)₃), 1.50–1.63 (m, 2H; OCH₂CH₂-

AHCTUNGTRENNUNG

AHCTNUGTREN(GUNN CH₂)₄CH₃), 4.85 (q, J=6.2 Hz, 1H; Trp CH), 5.19 (brs, 1H; NH), 6.60–

7.55 ppm (m, 7H; CONH and indole CH); ¹³C NMR: d=14.2, 22.7, 25.9,

27.6, 28.5, 28.6, 29.0, 31.8, 32.9, 43.0, 44.3, 53.4, 65.9, 80.5, 108.4, 109.6,

118.7, 119.3, 122.0, 127.9, 128.3, 137.1, 156.2, 168.5, 170.1, 171.9 ppm.

ACHTUNGTRENNUNG(C₁₈H₃₇)₂NCOOCH₂OCH₂CO-Gly-Gly-Gly-Pro-Gly-Gly-Trp-OC₇H₁₅

(12): Compound 13 (140 mg, 0.10 mmol) was dissolved in THF (7.5 mL).

NaOH solution (1.5 mL, 0.1m aq.) was added dropwise to stirred solu-

tion. The mixture was stirred at RT for 40 min. TLC (CHCl₃:MeOH=

90:10) indicated complete transformation. Acetic acid (glacial) was

added to adjust to pH 5–6 and the solvents were evaporated in vacuo.

The residue was purified by column chromatography (silica gel, CHCl₃/

MeOH=93:7). Recrystallization from MeOH afforded a white solid

(100 mg, 73%). M.p. 118–1208C; ¹H NMR (CDCl₃): d=0.80–1.00 (m,

Boc-PGGW

(N-CH₃)-OC₇H₁₅

:

Boc-GGW

(N-CH₃)-OC₇H₁₅

(0.22 g,

(N-

0.41 mmol) was deprotected in 4n HCl/dioxane for 2 h. HCl·GGWACHTUNGTRENNUNG

CH₃)-OC₇H₁₅, Boc-Pro-OH (0.089 g, 0.41 mmol), EDCI (0.087 g,

0.46 mmol), and HOBt (0.062 g, 0.46 mmol) were suspended in CH₂Cl₂

(25 mL) and Et₃N (0.17 mL) was added. The mixture was stirred at RT

for 24 h. Evaporation of the solvent and chromatography (silica gel,

CHCl₃/MeOH=98:2- 95:5) gave

a viscous semi-solid (0.26 g, 98%).

¹H NMR: d=0.89 (t, J=6.8 Hz, 3H; CH₂CH₃), 1.26 (pseudo-s, 8H;

9H; CH₂CH₃), 1.18–1.40 (m, 68H; NCH₂CH

(CH₂)₄CH₃), 1.49 (pseudo-s, 4H; NCH₂CH₂A

6.8 Hz, 2H; OCH₂CH₂A(CH₂)₄CH₃), 1.72–2.18 (m, 4H; Pro

NCH₂CH₂CH₂), 3.03 (brt, J=6.8 Hz, 2H; NCH₂CH₂A(CH₂)₁₅CH₃), 3.14–

3.34 (m, 4H; NCH₂CH₂A(CH₂)₁₅CH₃and Trp CH₂), 3.40–4.30 (m, 19H;

Pro NCH₂CH₂CH₂, Gly CH₂, Pro CH, OCH₂CH₂A(CH₂)₄CH₃and

OCH₂CO), 4.74 (brq, J=7.3 Hz, 1H; Trp CH), 7.00–7.20 (m, 3H; indole

CH), 7.30–7.60 (m, 4H; indole CH and CONH), 7.78–7.95 (m, 2H;

CONH), 8.01 (brs, 1H; CONH), 8.21 (bt, J=7.3 Hz, 1H; CONH),

9.53 ppm (s, 1H; indole NH); ¹³C NMR (CDCl₃): d=14.2, 14.3, 22.8,

22.9, 25.2, 26.0, 27.1, 27.3, 27.6, 27.8, 28.7, 29.0, 29.1, 29.2, 29.5, 29.6, 29.8,

29.9, 31.9, 32.1, 41.9, 42.9, 43.4, 46.6, 46.9, 47.1, 53.0, 61.3, 66.0, 69.6, 71.4,

109.4, 111.8, 118.2, 119.1, 121.7, 124.6, 127.4, 136.5, 168.7, 168.9, 170.3,

170.4, 170.5, 171.2, 172.5, 173.5 ppm; IR (CHCl₃): n˜ =3303, 3078, 2923,

2854, 1741, 1661, 1645, 1548, 1460, 1377, 1340, 1238, 1209, 1130,

₂A

OCH₂CH CHUTTGNERNN(UGN CH₂)₄CH₃), 1.43 (s, 9H; CAHCTNUGTRENGN(UN CH₃)₃), 1.48–1.61 (m, 2H;

₂A

G

CHTUNGTRENNUNG

OCH₂CH₂ACHTUNGTRNEN(NUG CH₂)₄CH₃), 1.82–2.20 (m, 4H; Pro NCH₂CH₂CH₂), 3.28 (d,

CTHUNGTRENNUNG

J=5.7 Hz, 2H; Trp CH₂), 3.34–3.55 (m, 2H; Pro NCH₂CH₂CH₂), 3.68–

4.20 (m, 10H; Gly CH₂, indole NCH₃, OCH₂CH₂A(CH₂)₄CH₃and Pro

CTHUNGTRENNUNG

CH), 4.83 (q, J=6.2 Hz, 1H; Trp CH), 6.73–7.65 ppm (m, 8H; CONH

and indole CH); ¹³C NMR: d=14.2, 22.8, 24.9, 25.9, 27.6, 28.6, 29.1, 29.6,

29.9, 31.8, 32.9, 43.3, 47.5, 53.3, 60.9, 65.8, 81.0, 108.6, 109.5, 118.8, 119.2,

121.8, 128.1, 128.4, 137.0, 155.9, 168.8, 169.9, 171.9, 173.5 ppm.

CTHUNGTRENNUNG

A

ACHTUNGRTEN(NUNG N-CH₃)-

AHCTUNGTRENNUNG

(C₁₈H₃₇)₂NCOCH₂OCH₂CO-GGG-OH (0.32 g, 0.40 mmol), EDCI

(0.084 g, 0.44 mmol), and HOBt (0.059 g, 0.44 mmol) were suspended in

CH₂Cl₂(20 mL). The mixture was cooled (08C) and Et₃N (0.17 mL) was

added. The mixture was stirred at RT for 60 h. The solvent was evaporat-

&

14

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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 0000, 00, 0 – 0

ÝÝ

These are not the final page numbers!

Anion Transport

FULL PAPER

ed, the residue was crystallized (MeOH), and then subjected to chroma-

tography (silica gel, CHCl₃/MeOH=93:7–90:10). Crystallization from

MeOH gave an off-white solid (0.27 g, 51%). ¹H NMR: d=0.88 (t, J=

Instruments), sampled at the 100 Hz of amplifier filter frequency and col-

lected by Clampex 9.2 (software from Axon Instruments). The data were

analyzed later using Clampfit v. 9.2 (software from Axon Instruments).

7.0 Hz, 9H; CH₂CH₃), 1.05–1.60 (m, 74H; NCH

(CH₂)₅CH₃), 1.90–2.23 (m, 4H; Pro NCH₂CH₂CH₂), 3.98–3.34 (m, 6H;

NCH₂CH₂A(CH₂)₁₅CH₃and Trp CH₂), 3.42–3.70 (m, 2H; Pro

NCH₂CH₂CH₂), 3.72–4.18 (m, 17H; indole NCH₃, Gly CH₂, OCH₂CH₂-

(CH₂)₄CH₃and OCH₂CO), 4.20–4.37 (m, 3H; Pro CH and OCH₂CO),

₂ACHTGNUERTN(NUNG CH₂)₁₆CH₃and OCH₂-

ACHTUNGTRENNUNG

CHTUNGTRENNUNG

Acknowledgements

ACHTUNGTRENNUNG

4.62 (q, J=6.3 Hz, 1H; Trp CH), 7.00–7.15 (m, 2H; indole CH), 7.17–

7.30 (m, 3H; indole CH and CONH), 7.45–7.60 (m, 2H; indole CH and

CONH), 7.86–8.03 (m, 3H; CONH), 8.39 ppm (brt, J=7.3 Hz, 1H;

CONH); ¹³C NMR: d=14.2, 14.3, 22.8, 22.9, 25.4, 25.9, 27.1, 27.3, 27.4,

27.8, 28.6, 29.0, 29.1, 29.2, 29.5, 29.6, 29.7, 29.8, 29.9, 31.9, 32.8, 42.1, 43.0,

43.1, 43.2, 43.7, 46.7, 47.1, 47.2, 53.8, 61.5, 65.9, 69.7. 71.6, 108.8, 109.5,

118.6, 119.1, 121.7, 128.0, 128.4, 137.1, 168.9, 169.1, 170.1, 170.2, 170.4,

170.5, 171.5, 172.6, 173.5 ppm. elemental analysis calcd (%) for

C₇₄H₁₂₇N₉O₁₁: C 67.39, H 9.71, N 9.56; found: C 67.05, H 9.47, N 9.38.

We thank the NIH for a grant (GM-63190) and the NSF (CHE-0957535)

for grants that supported this work. We also gratefully acknowledge the

expertise of Dr. Dan Zhou and the resources of the MIST lab, Center for

Nanoscience.

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Published online: && &&, 0000

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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 0000, 00, 0 – 0

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These are not the final page numbers!

Anion Transport

FULL PAPER

Self-assembly versus dynamics: The

work presented addresses the relation-

ship of self-assembly and aggregation

to the efficacy of Cl^ꢀtransport. A

range of synthetic anion transporters

(SATs) was prepared and their ability

to transport Cl^ꢀthrough liposomal

membranes was confirmed. The gen-

eral conclusion from these studies was

that those compounds that self-aggre-

gated were generally poor ion trans-

porters. (DOPC/DOPA=1,2-dioleoyl-

sn-glycero-3-phosphocholine/phos-

phate).

Anion Transport

M. M. Daschbach, S. Negin, L. You,

M. Walsh, G. W. Gokel* . . . . &&&& — &&&&

Aggregation and Supramolecular

Membrane Interactions that Influence

Anion Transport in Tryptophan-

Containing Synthetic Peptides

Chem. Eur. J. 2012, 00, 0 – 0

ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

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ÞÞ

These are not the final page numbers!

Article Doi

Aggregation and supramolecular membrane interactions that influence anion transport in tryptophan-containing synthetic peptides

DOI: 10.1002/chem.201103203

Source and publish data:

Authors:

Article abstract of DOI:10.1002/chem.201103203

Full text of DOI:10.1002/chem.201103203

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