Supporting Information © Wiley-VCH 2005

69451 Weinheim, Germany

Supplementary Information

for

The Internal Structure of Helical Pores Self-Assembled from Dendritic Dipeptides is Stereochemically

Programmed and Allosterically Regulated

Virgil Percec,* Andrés E. Dulcey, Mihai Peterca, Monica Ilies, Janine Ladislaw, Brad M. Rosen, Ulrica

Edlund, Paul A. Heiney

[*] Prof. V. Percec, A. Dulcey, Dr. M. Ilies, J. Ladislaw, B. M. Rosen, Dr. U. Edlund

Roy & Diana Vagelos Laboratories, Department of Chemistry

Laboratory for Research on the Structure of Matter

University of Pennsylvania

Philadelphia, PA 19104-6323 (USA)

Fax: (+1) 215-573-7888

E-mail: percec@sas.upenn.edu

M. Peterca, Prof. P. A. Heiney

Department of Physics and Astronomy

Laboratory for Research on the Structure of Matter

University of Pennsylvania

Philadelphia, PA 19104-6396 (USA)

Table of Contents

S1. Materials

S2. Techniques

S3. Synthesis of Dendritic Dipeptides

S4. Analysis of Self-Assembly by CD and UV

Figure SF1. CD and UV spectra of dendritic dipeptides

Figure SF2. CD and UV spectra of model compounds for dendritic dipeptides

Scheme SF1. Model compounds and precursors for dendritic dipeptides

Figure SF3. UV spectra of molecular solutions of dendritic dipeptides and their precursors

Figure SF4. CD and UV spectra of dendritic dipeptides and model compounds at a single temperature

S5. Analysis of Supramolecular Structures by X-ray Diffraction Experiments

Figure SF5. Temperature dependence of X-ray diffraction stack plots for (4-3,4-3,5)12G2-CH2-Boc-L-

Tyr-L-Ala-OMe and for (4-3,4-3,5)12G2-CH2-Boc-D-Tyr-D-Ala-OMe

Figure SF6. Temperature dependence of X-ray diffraction stack plots for (4-3,4-3,5)12G2-CH2-Boc-L-

Tyr-D-Ala-OMe and for (4-3,4-3,5)12G2-CH2-Boc-D-Tyr-L-Ala-OMe

Figure SF7. Detailed view of the stereochemistry induced change in dipeptide conformation

S6. Determination of the Dependence of Dcol and Dpore on Temperature

Table ST1. X-ray diffraction data of dendritic dipeptides

Figure SF8. H-bonding network for L-L, D-D, L-D and D-L dendritic dipeptides and the formation of

the supramolecular pore structure

S7. References

2

S1. Materials

Methyl 4-hydroxybenzoate (99%) (from Lancaster Synthesis), thionyl chloride (99.5%), LiAlH4 (95%),

N-methyl morpholine (99%), methyl chloroformate (98%), anhydrous K2CO3 (all from Aldrich), 3,4-

dihydroxybenzoic acid (97%), 3,5-dihydroxybenzoic acid (97%), 1-bromododecane (98%), tyrosine (99%),

cyanuric chloride (99%), triphenylphosphine (99%) (all from Acros Organics), Boc-L-Tyr-OH (99%, [α]D24 = +

37.9o, 1% in dioxane), Boc-D-Tyr-OH (99%, [α]D24 = - 38.3o, 1% in dioxane), Boc-DL-Tyr-OH (99%), H2N-L-

Ala-OMe.HCl (99%, [α]D24 = + 7.2o, 2% in methanol), H2N-D-Ala-OMe.HCl (99%, [α]D

24 = - 7.5o, 2% in

methanol), H2N-DL-Ala-OMe.HCl (99%), (all from Bachem Peptides) were used as received. Acetone, N,N-

dimethylformamide, ethyl acetate, magnesium sulphate, methanol (all from Fisher, ACS reagents), silica gel

(Sorbent Technology) were used as received. Tetrahydrofuran (THF) and dichloromethane (Fisher, ACS reagent

grade) were refluxed over sodium/benzophenone and CaH2, respectively, and freshly distilled before use.

Cyclohexane and trifluoroethanol for CD experiments, dichloromethane for HPLC assays and THF for MALDI-

TOF assignments and for UV spectra were HPLC grade (Fisher). Deuterated chloroform for NMR spectra was

from Cambridge Isotope Laboratories. All other chemicals were commercially available and were used as

received. 2,6–Di-tert-butyl-4-methylpyridine (DTBMP) was prepared using a literature procedure[1] and 2-

chloro-4,6-dimethoxy-1,3,5-triazene (CDMT) was obtained from cyanuric chloride following a literature

synthesis.[2]

S2. Techniques

The purity of the products was determined by a combination of thin-layer chromatography (TLC), high

pressure liquid chromatography (HPLC), 1H and 13C NMR, as well as by matrix-assisted laser

desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. TLC was carried on silica gel coated

aluminum plates (indicator, F254; layer thickness, 200 µm; particle size, 2-25 µm; pore size 60Å, SIGMA-

Aldrich). HPLC was performed in dichloromethane, on a Perkin-Elmer Series 10 high pressure liquid

chromatograph equipped with a LC-100 column oven, Nelson Analytical 900 Series integrator data station and

two Perkin-Elmer PL gel columns of 5 x 102 and 1 x 104 Å, using for detection the UV absorbance at 254 nm. 1H

NMR (500 MHz) and 13C NMR (125 MHz) spectra were recorded on a Bruker DRX 500 instrument.

3

UV spectra were registered on a Shimadzu-1601 UV spectrophotometer at 23 oC, in THF (1.6 x 10-5 M)

using a quartz cuvette of 0.1 cm path length.

Thermal transitions were measured on a Thermal Analysis (TA) Instrument modulated differential

scanning calorimeter (DSCQ100). In all cases the heating and the cooling rates were 10 °C min-1. The transition

temperatures were measured as the maxima and minima of their endothermic and exothermic peaks. Indium was

used as calibration standard. An Olympus BX51 thermal optical microscope (100X magnification) equipped

with a Mettler FP82HT hot stage and a Mettler Toledo FP90 Central Processor were used to verify thermal

transitions and to characterize anisotropic textures. Small parts of the aligned samples were heated on the hot

stage up to temperatures close but always bellow isotropic transition to avoid losing the sample alignment. After

a short annealing, image acquisitions were performed. Density measurements were carried out by floatation in

gradient columns at 20 oC.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was carried

out on a PerSeptive Biosystems-Voyager-DE (Framingham, MA) mass spectrometer operating in linear mode.

The spectrometer equipped with a nitrogen laser (337 nm) was calibrated using Angiotensin II and Bombesin as

standards. The laser steps and voltages applied were adjusted depending on both the molecular weight and the

nature of each analysed compound. Either 2,5-dihydroxybenzoic acid or 4-hydroxybenzylidenemalononitrile was

used as matrix. THF was used for dissolving both matrix and sample (working concentrations: 10 mg/ml for the

matrix and 5-10 mg/ml for the sample). The analytical sample was obtained my mixing sample and matrix

solutions in a 1/5 v/v ratio; 0.5 µL of this resulting solution were loaded onto the MALDI plate and allowed to

dry at 23 oC before performing the analysis.

Circular dichroism (CD) spectra were recorded on a Jasco J-720 spectrophotometer equipped with a

NESLAB RTE-111 variable temperature circulator. Each sample of dendritic dipeptides was dissolved in

cyclohexane (1.6 x 10-4 M) at 23 oC, then heated to 60 oC and allowed to cool at 23 oC when the solution

remained homogenous. (4-345)12G1-CO2-L-DEG* and (4-345)12G1-CO2-D-DEG* used as model compounds

were dissolved in trifluoroethanol (3 x 10-4 M). CD measurements were performed using a 1 ml quartz cuvette of

0.1 cm path length and the following parameters: scanned optical range, 210-320 nm; scan band width, 1 nm;

scanning speed, 100 nm/min; response, 1 sec; accumulations, 5; 3 scanned thermal ranges: 8-60 oC, 60-8 oC, 8-

4

60 oC, respectively (data pitch: 2 oC; temperature slope: 1 oC/min). Before starting the experiment the sample

was allowed to reach the 8 oC starting temperature (~ 10-15 minutes) but once started the 3 thermal cycles were

performed successively. Data were processed using Jasco Spectra Manager V. 1.51 software.

X-ray diffraction (XRD) measurements were performed using Cu-Kα1 radiation (λ=1.54178 Å) from a

Bruker-Nonius FR-591 rotating anode X-ray source with a 0.2 x 0.2 mm2 filament operated at 3.4 kW. The beam

was collimated and focused by a single bent mirror and sagitally focusing Si (111) monochromator, resulting in a

0.3 x 0.4 mm2 spot on a Bruker-AXS Hi-Star multiwire area detector. To minimize attenuation and background

scattering, an integral vacuum was maintained along the length of the flight tube and within the sample chamber.

Powder or aligned samples were held in quartz capillaries (0.7 – 1.0 mm diameter), than mounted in a

temperature controlled oven (temperature precision: ± 0.1 oC, temperature range from -120°C to 270°C). Sample

to Bruker-AXS Hi-Star multiwire area detector distances are 12.0 cm for wide angles diffraction experiments,

and 54.0 cm for intermediate angles diffraction experiments. Aligned samples for fiber XRD experiments were

prepared using a custom made extrusion device. The powdered sample (~ 10 mg) was heated inside the extrusion

device to isotropic transition temperature. After cooling slowly from the isotropic phase, the fiber was extruded

in the mesophase and cooled to 23 oC. Typically the aligned samples have a thickness of ~ 0.3-0.7 mm and a

length of ~ 3-7 mm. All XRD measurements were done with the aligned sample axis perpendicular to the beam

direction. XRD peaks position and intensity analysis was performed using Datasqueeze Software (version 2.01)

that allows background elimination and Gaussian, Lorentzian, Lorentzian squared, or Voigt peak-shape fitting.

Molecular modelling and simulation experiments were performed using the Materials Studio Modelling

(version 3.1) software from Accelrys. Package’s Discover module was used to perform the energy minimizations

on the supramolecular structures with the following settings: PCFF or COMPASS force fields, and Flechter-

Reeves algorithm for the conjugate gradient method. Details were repoted previously.[3] The reported molecular

models are in agreement with the small and wide angle XRD results on oriented fibers and with the experimental

densities results.[3]

S3. Synthesis of Dendritic Dipeptides

Synthesis of dendritic dipeptides was carried out as previously described.[3] All samples were purified by

flash column chromatography (silica gel, eluent MeOH/CH2Cl2 1%) until the purity was higher than 99%,

5

followed by precipitation into methanol from a concentrated solution in CH2Cl2. The sample was filtered and

dried to constant weight under vacuum at 23 oC. The complete structural analysis for the newly synthesized

derivatives was also reported in the same paper.[3] The measured densities of the supramolecular structures

generated from dendritic dipeptides are between 0.99 – 1.02 g/cm3.

S4. Analysis of Self-Assembly by CD and UV

CD and UV spectra for all dendritic dipeptides during their self-assembly in cyclohexane are presented

in Figure SF1. Figure SF2 shows the self-assembly of a self-assembling benzyl ether dendritic molecule (4-

3,4,5)12G1-CO2-L and D-DEG* (Scheme SF1) that contains an aliphatic stereocenter attached to the dendron via

an ester group. Even if self-assembly of these two sets of moleculaes takes place in different solvophobic

solvents, comparison of the CD spectra from Figures SF1a,b and SF2a,b indicates that the CD spectra of L-L, D-

D, L-D and D-L are due to the aromatic part of the dendron. The CDs of the dipeptides and of the dendritic

dipeptides as molecular solutions show a weak Cotton effect at 232 nm that has an opposite sign to that of the

Cotton effects of the self-assembled dendrons from the same range of the spectrum (Figure SF1). The molecular

Cotton effect can be obtained in good solvent or in solvophobic solvents above 30oC.[3] The Cotton effect from

above 290 nm of the (4-3,4,5)12G1-CO2-L and D-DEG* (Figure SF2) is due to the benzoate moiety of the (4-

3,4,5)12G1-CO2-DEG* dendron (Figure SF3).

6

7

8

9

10

11

12

The chemical structures of the compounds analyzed by UV spectra are shown in Scheme SF1.

HN

O

NH

OCH3

O

O

O

OH

HN

O

NH

OCH3

O

O

O

OH

HN

O

NH

OCH3

O

O

O

OH

HN

O

NH

OCH3

O

O

O

OH

HO HOOC12H25

HOO

O

OC12H25

OC12H25 HO

O

O

O

O

O

O

OC12H25

OC12H25

OC12H25

OC12H25

4-12G0-CH2OH C6H5-CH2-OH

Boc-D-Tyr-D-Ala-OMe Boc-L-Tyr-L-Ala-OMe

(4-3,4)12G1-CH2OH

Boc-L-Tyr-D-Ala-OMe Boc-D-Tyr-L-Ala-OMe (4-3,4-3,5)12G2-CH2OH

O

OO

OC12H25

OC12H25

OC12H25

OH3C

OO

OO

OC12H25

OC12H25

OC12H25

HO

OO

OO

OC12H25

OC12H25

OC12H25

HO

(4-3,4,5)12G1-COOCH3 (4-3,4,5)12G1-COOH (4-3,4,5)12G1-CH2OH

O

OO

OC12H25

OC12H25

OC12H25

O

OO

HO O

OO

OC12H25

OC12H25

OC12H25

O

OO

HO

(4-3,4,5)12G1-CO2- D-DEG* (4-3,4,5)12G1-CO2- L-DEG*

Scheme SF1. Model compounds and precursors for the dendritic dipeptides.

As shown in Figure SF3a, the higher the dendrimer generation the more intense the UV absorption, as

one should expect as a result of the increasing number of aromatic units in the structure. Figure SF3b illustrates

the UV absorption of the dendritic dipeptides as compared to the UV absorption of the dipeptides, whereas

Figure SF3c presents UV spectra for a dendron containing at its apex other structural moieties than the

dipeptides and for its precursors. The UV spectra in Figure SF3a,b demonstrate that the aromatic part of the

dendron absorbs below 290 nm. The addition of a carboxylic unit to the aromatic part of the dendron extends the

absorption above 290 nm (Figure SF3c).

13

220 230 240 250 260 270 280 290 300 310 3200.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

Abs

λ /nm

Benzyl Alcohol (4)-12G0CH2OH (4-3,4)12G1CH2OH (4-3,4-3,5)12G2CH2OH (4-3,4-3,5)12G2CH2Boc-D-Tyr-D-Ala-OMe (4-3,4-3,5)12G2CH2Boc-D-Tyr-L-Ala-OMe (4-3,4-3,5)12G2CH2Boc-L-Tyr-D-Ala-OMe (4-3,4-3,5)12G2CH2Boc-L-Tyr-L-Ala-OMe

in THF (1.6 x 10-5 M)

220 230 240 250 260 270 280 290 300 310 3200.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Figure SF3. UV spectra of molecular solutions at 23°C: a) absorption increases with the increase of dendrimer generation,

i.e. increase number of aromatic units; b) absorption of dendritic dipeptide compared with dipeptide absorption; c)

absorption of (4-3,4,5)12G1 dendron containing different groups at the apex.

Figure SF4 compares the CD-UV spectra of (4-3,4-3,5)12G2-CH2-Boc-L-Tyr-L-Ala-OMe and of (4-

3,4,5)12G1-CO2-L-DEG* ( both in cyclohexane) at the temperature at which their CD signal was most intense.

As it can be seen, below 290 nm both spectra present similar patterns. The UV absorption and the CD signal of

(4-3,4,5)12G1-CO2-L and D-DEG* above 290 nm are due to its benzoate ester part.

1.8

in THF (1.6 x 10-5 M)

(4-3,4-3,5)12G2-CH2-Boc-L-Tyr-L-Ala-OMe Boc-D-Tyr-D-Ala-OMe Boc-D-Tyr-L-Ala-OMe Boc-L-Tyr-D-Ala-OMe Boc-L-Tyr-L-Ala-OMe

Abs

λ /nm

220 230 240 250 260 270 280 290 300 310 320 330 340 350

b) a)

c)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

in THF (1.6 x 10-5 M)

λ /nm

Abs

(4-3,4,5) 12G1-CH2OH

(4-3,4,5) 12G1-COOCH3

(4-3,4,5) 12G1-COOH (4-3,4,5) 12G1-CO2-D-DEG*

14

220 240 260 280 300 320 3400.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

220 240 260 280 300 320 340

-200-180-160-140-120-100-80-60-40-20020406080

Θ x10-3/degcm2dmol-1

Abs

λ /nm

(4-3,4-3,5)12G2-CH2-Boc-L-Tyr-L-Ala-OMe in cyclohexane at 8°C (1.6 x 10-4 M)

UV

CD

220 240 260 280 300 320 3400.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

220 240 260 280 300 320 340

-140-120-100-80-60-40-20

020406080

Θ x10-3/degcm2dmol-1

Abs

λ /nm

(4-3,4,5)12G1-CO2-L-DEG* in trifluoroethanol at 10°C (3.0 x 10-4 M)

CD

UV

a)

b)

Figure SF4. Combined CD-UV spectra of: a) (4-3,4-3,5)12G2-CH2-Boc-L-Tyr-L-Ala-OMe in cyclohexane (1.6 x 10-4 M)

and b) (4-3,4,5)12G1-CO2-L-DEG* in trifluoroethanol (3 x 10-4 M).

15

S5. Analysis of Supramolecular Structures by X-ray Diffraction Experiments

Supporting X-ray data are shown in Figures SF5 and SF6, while Figure SF7 shows a detailed view of the

dipeptide and dendron conformations.

(10) (10)

I (11) (11) / a.u. (20) I (20)

/ a.u.(21) (21)

(10) (10)

I (11) (11) / a.u.(20) I (20) / a.u.

(21) (21)

Figure SF5. Stacked plots showing temperature dependence of X-ray diffraction for (4-3,4-3,5)12G2-CH2-Boc-L-Tyr-L-

Ala-OMe (top): second heating (left), second cooling (right) and for (4-3,4-3,5)12G2-CH2-Boc-D-Tyr-D-Ala-OMe (bottom):

second heating (left), second cooling (right).

16

(10) (10)

I I (11) / a.u.(11)

(20) / a.u.(20) (21) (21)

(10) (10)

I (11) I (11) (20) / a.u. / a.u.(20) (21) (21)

Figure SF6. Stacked plots showing temperature dependence of X-ray diffraction for (4-3,4-3,5)12G2-CH2-Boc-L-Tyr-D-

Ala-OMe (top): second heating (left), second cooling (right) and for (4-3,4-3,5)12G2-CH2-Boc-D-Tyr-L-Ala-OMe (bottom):

second heating (left), second cooling (right).

17

L- L D-D

L-D D-L

Figure SF7. Detailed view of the stereochemistry induced change in dipeptide conformation: for simplicity the (4-3,4-

3,5)12G2-CH2-dendron is shown without the aliphatic tails, dashed squares indicate Tyr stereocenter that dictates the

supramolecular helix sense.

S6. Determination of the Dependence of Dcol and Dpore on Temperature

The dependence of Dcol and Dpore on temperature was obtained from powder small angle X-ray

diffraction experiments performed at various temperatures (see Table ST1). Dcol was calculated from the X-ray

diffractograms data by averaging the lattice parameter a calculated from (10), (11), and (20) peaks position: Dcol

= a = {4*π/[3*sqrt(3)]}*(1/q10+sqrt(3)/q11+2/q20) Å, where qhk is the scattering vector for the (hk) reflection.

Dpore was calculated from the integrated (hk0) peaks intensities using the method reported in detail in the

Supplementary Material to ref. [3] and in reference [4]. The X-ray diffractograms used for these calculations are

presented in Figures SF5 and SF6.

Figure SF8 presents in detail H-bonding network (lengths in Å) for L-L, D-D, L-D and D-L dendritic

dipeptides and the formation of the supramolecular pore structure.

18

Table ST1: X-ray diffraction data collected at 20°C.

Compound T [°C]

d10a [Å]

(I10b a.u.)

d11a [Å]

(I11b a.u.)

d20a [Å]

(I20b a.u.)

d21a [Å]

(I21b a.u.)

a=Dcol[Å]

Dpore [Å]

(4-3,4-3,5) 12G2 CH2 Boc -

L-Tyr-L-Ala 20 67.0 (43.72)

38.4 (26.63)

33.5 (24.27)

25.5 (5.36) 77.1±0.4 13.3±1.2

D-Tyr-D-Ala 20 68.5 (43.25)

39.4 (26.0)

34.2 (24.23)

26.1 (6.51) 78.9±0.4 13.9±1.2

L-Tyr-D-Ala 20 69.7 (42.01)

40.0 (26.46)

34.7 (25.0)

27.4 (5.37) 80.2±0.4 14.6±1.4

D-Tyr-L-Ala 20 69.4 (40.7)

39.8 (26.61)

34.4 (23.5)

27.2 (9.17) 79.8±0.4 15.4±1.4

[a] columnar hexagonal phase d-spacings; [b] peak intensity scaled to the sum of the observed diffraction peaks (a.u.=arbitrary units).

Figure SF8. H-bonding network (lengths in Å) for a) L-L, b) D-D, c) L-D and d) D-L dendritic dipeptides and the formation

of the supramolecular pore structure. The strongest in layer directional H-bonds are labeled by the same convention as in

Figure 5: j-k and l-m, respectively. Column axis is vertical; for simplicity only the N-hydrogen atoms are shown (colored in

white in a), b), c), d) and green in e), f), g) and h), while the carbon atoms of each dipeptide are shown in different colors

(same color for one dipeptide) and the phenyl ring of Tyr is omitted. The helical H-bonded supramolecular structure

resulted from the j-k and l-m hydrogen bonds:e) and f) - side view and g) - top view. Complete pore structure in h) - side

view and i) - top view (dendron is not shown, color code: C - gray, H – white, N-H – green, Boc – blue, O – red).

D-L

2.21

2.04 1.31 1.32

2.21

2.15

1.16

2.04

k j

l m

m L-L

2.33

2.09

1.88

1.96

1.79

2.3

j k

j

m

k

l

L-D

2.23

2.33 1.1

2.2

2.16

1.28

2.46

1.15 j k

m

l

2.3

e) f) a) b) h) D-D

l

2.43 2.43 2.12

2.21 2.12

k j

d) c)

k i) g)

k j

m j

l

19

S7. References. [1] P.J. Stang, A.G. Anderson, J. Org. Chem. 1976, 41, 3034-3036.

[2] J.S. Kronin, F.O. Ginah, A.R. Murray, J.D. Copp, Synthetic Commun. 1996, 26,3491-3494

[3] V. Percec, A.E. Dulcey, V.S.K. Balagurusamy, Y. Miura, J. Smidrkal, M. Peterca, S. Nummelin, U. Edlund,

S.D. Hudson, P.A. Heiney, H. Duan, S.N. Maganov, S.A. Vinogradov Nature 2004, 430, 764-768.

[4] D.C. Turner, S.M. Gruner, Biochemistry 1992, 31, 1340-1355.

20