Supporting Information © Wiley-VCH 2006

69451 Weinheim, Germany

1

Mapping the Landscape of Potentially Primordial Informational Oligomers:

Oligo-dipeptides and Oligo-dipeptoids Tagged with Triazines as Recognition

Elements**

Gopi Kumar Mittapalli, Kondreddi Ravinder Reddy, Hui Xiong, Omar Munoz, Bo Han,

Francesco De Riccardis, Ramanarayanan Krishnamurthy* and Albert Eschenmoser*

______________________________________________________________ [*] Prof. Dr. A. Eschenmoser, Dr. B. Han

Laboratorium für Organische Chemie Eidgenössische Technische Hochschule HCI Hönggerberg CH-8093 Zürich Switzerland Fax: (+41)1-632-1043 E-mail: Eschenmoser@org.chem.ethz.ch

Prof. Dr. A. Eschenmoser, Prof. Dr. R. Krishnamurthy, Dr. G. K. Mittapalli, Dr. K. R. Reddy, Dr. H. Xiong, Dr. O. Munoz, Dr. F. De Riccardis Department of Chemistry and The Skaggs Institute for Chemical Biology The Scripps Research Institute 10550 North Torrey Pines Road, La Jolla, CA 92037 Fax: (+1)858-784-9573 E-mail: rkrishna@scripps.edu

[**] Chemistry of α-aminonitriles Part 45. For part 44 see [1a] and for part 43 see [1b]. This work was

supported by the Skaggs Research Foundation. G. K. M., K. R. R., H. X., O. M., and F. D. R. are Skaggs Postdoctoral Fellows.

2

Experimental Part

General. Solvents for extraction: ACS grade. Solvents for reaction: reagent grade. Reagents:

unless otherwise noted, from Acros, Fluka, or Aldrich, highest quality available. TLC: silica gel

60 F254 aluminum plates, (Whatman, type Al Sil G/UV, 250 µm layer); visualization by UV

absorption) and/or by dipping in a soln. anisaldehyde/H2SO4/AcOH/EtOH 5:5:1:18)

cerium(IV)sulfate (3 mM)/ammonium molybdate (250 mM) in aq. H2SO4 (10%); followed by

heating. Flash column chromatography (CC) was performed on silica gel 60 (0.40 – 0.63 mm,

230 – 440 mesh, EM Science) at low pressure (max. 2 bar). Melting points (uncorrected) MEL-

TEMP II (Laboratory Devices Inc., USA). NMR: 1H: δ values in ppm (TMS as internal

standard); J [Hz], assignments of 1H resonances were in some cases based on 2D experiments

(1H,1H-COSY); 13C: δ values in ppm (TMS as internal standard); J [Hz]; assignments and

multiplicities were based on 2D experiments (1H,13C-COSY). ESI-MS (mode): m/z (intensity in

%); performed with Micromat-LCT. Matrix-assisted laser-desorption-ionization time-of-flight

mass spectrometry (MALDI-TOF-MS) was performed on a Voyager-Elite mass spectrometer

(Perseptive Biosystems) with delayed extraction with THAP or DHB as the matrix with

ammonium citrate added to the sample. Oligopeptides were synthesized on an Expedite 8909

Nucleic Acid Synthesis system (Perseptive Biosystems) in the PNA mode with the following

modifications: 0.14M dipeptide monomer in NMP/DMSO 4:1; 0.13M of HATU in NMP;

washing soln. A: NMP/DMSO 4:1; washing soln. B: NMP/DMSO 4:1; deblocking soln. 30%

piperidine in NMP/DMSO 4:1; capping: 5% Ac2O, 6% lutidine in NMP/DMSO 4:1; base-

mixture: 0.14M DIPEA, 0.21M lutidine in NMP; coupling time of 30 min; in some cases double

coupling step was used. Coupling efficiency was calculated by Fmoc-assay. For a majority of the

synthesis, the average coupling efficiency was greater than 95%. HPLC purification of

oligopeptides was achieved either by (a) ion-exchange: with MONO-Q HR 5/5 Pharmacia, 10 Ï

0.5 cm or Nucleogen-DEAE 60-7 Machery Nagel, 125 Ï 4, flow 1 ml / min; mobile phase:

eluant A: 10 mM Na2HPO4, H2O, pH 11.5; eluant B: 10 mM Na2HPO4, 1 M NaCl, H2O, pH

11.5; or with Nucleogen-DEAE 60-7 Machery Nagel, 125 Ï 4, flow 1 ml / min, mobile phase:

eluant A: 10 mM Na2HPO4, H2O, pH 7.0; eluant B: 10 mM Na2HPO4, 1 M NaCl, H2O, pH 7.0 or

by (b) reverse-phase: Aquapore ODS 20 micron Brownlee, 250 Ï 10.0 mm, flow 4 ml / min.

Mobile phase: eluant A: 0.1% TFA in H2O; eluant B: 0.1% TFA in MeCN. UV Spectra were

recorded on a Cary 1 C spectrophotometer (Varian). Melting point (Tm) measurements of

3

oligonucleotides were determined with Cary 1 Bio spectrophotometer (Varian). CD Spectrum

was measured on an AVIV 61 DS CD spectropolarimeter. All measurements were made with the

′phosphate buffer′, 10 mM aq. NaH2PO4 buffer containing 0.1 mM Na2EDTA, 150 mM (or 1M)

NaCl at pH 7.0, with a total oligonucleotide concentration of ca. 10 µM, unless indicated

otherwise, and the samples were thoroughly degassed, either by heating or by vacuum and

ultrasonication. The following molar extinction coefficients were used for the heterocyclic bases:

ε260(uracil) = 10,000, ε260 (thymine) = 10,000, ε260 (adenine) = 15,000, ε260 (2,4-diaminopurine)

= 10,000, ε260 (2,4-diaminotriazine) = 4200, ε250 (2,4-dioxotriazine) = 7500. Abbreviations: Asp

= aspartic acid; Bn = Benzyl; Boc = tert-butyloxy carbonyl; CBz = carboxybenzyl; DBU =

diazabicyclo undecane; DIPEA: diisopropylethylamine; EDCI= 1-ethyl-3-(3’-

dimethylaminopropyl)carbodiimide; Fmoc= 9-fluorenylmethyloxycarbonyl; Glu =

glutamic acid; HATU = 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium

hexafluorophosphate methanaminium; HOBt = 1-hydroxy benzotriazole; MBHA resin =

4-methylbenzhydrylamine resin; NMP = N-methyl-2-pyrrolidone.

4

Boc-HN COOBn

COOH

1) 1.1 equiv. (0.2M) DCC 1.1 (0.2) HOBt dry DMF, r.t., 12h

2) 2.0 (0.2M) biguanide DMF, r.t., 2h

61%

Boc-HN COOBn

N

N

N

NH2H2N

2) 1.5 (0.4) Fmoc-Cl 5 DIPEA, DMF, r.t., 12h

83%

1) 0.25M TFA in CH2Cl2 r.t., 2h

Fmoc-HN COOBn

N

N

N

NH2H2N

Fmoc-HN COOH

N

N

N

NH2H2N

10% Pd-CDMF/1,4-cyclohexadiene (5:1)r.t., 5h

64%

1) 0.25M TFA in CH2Cl2, r.t. , 2h

2) 1.0 (0.14) Fmoc-Asp(OtBu)-OH 1.2 (0.16) EDCI,1.2 (0.16) HOBt 1.0 (0.12) DIPEA, DMF/CH2Cl2 (1:1) r.t., 5h

88%

HN COOBn

N

N

N

NH2H2N

OFmoc-HN

t BuOOCHN COOH

N

N

N

NH2H2N

OFmoc-HN

t BuOOC10% Pd-C DMF/1,4-cyclohexadiene (5:1)r.t., 2h

66%

s1 s2 s3 s4

s5 s6

Scheme 1. Synthesis of the 2,4-diaminotriazine tagged Glu-Asp dipeptide monomer building block s6 used in the solid support synthesis of oligomer containing backbone type A (see Figure 1 of the manuscript).

HH

H HHH

H H

5

Boc-HN COOH

CONH2

81%

Boc-HN COOBn

NH

NH

N

OO

1) 0.25M TFA in CH2Cl2, r.t. , 2h

2) 1.0 (0.12) Fmoc-Asp(OtBu)-OH 1.2 (0.15) EDCI,1.2 (0.15) HOBt 1.0 (0.12) DIPEA, DMF/CH2Cl2 (1:1) r.t., 5h

83%

HN COOBn

NH

NH

N

OO

OFmoc-HN

t BuOOC

10% Pd-C DMF/1,4-cyclohexadiene(5:1)5-10?C, 6h

81%

s7

s12

Boc-HN COOBn

CONH2

77%

s8

2 equiv. (0.4M) NaHCO35 (1) PhCH2Br

DMF, r.t., 48h

1) 0.5 (0.1) Lawessson's reagent dry THF, r.t., 12h

Boc-HN COOBn

C(NH)SMe

s92) 10 (3) MeI, CH2Cl2, r.t., 12h3) 1 (7.5) aq. K2CO3, r.t., 10 min.

98%

Boc-HN COOBn

C(NH)NH2?HCl

s10

1 (0.2) NH4Cldry MeOH, reflux, 3h

86%

2.0 (0.4) PhOOCNHCOOPh2.0 (0.4) DBU, dry CH2Cl2, r.t., 2h

62%

s11

HN COOH

NH

NH

N

OO

OFmoc-HN

t BuOOC

s13

H H H

HHHH

H H

Scheme 2. Synthesis of the 2,4-dioxotriazine tagged Glu-Asp dipeptide monomer building block s13 used in the solid support synthesis of oligomer containing backbone type A (see Figure 1 of the manuscript).

6

Boc-HN

HOOC

COOBn1) 1.1 equiv. (0.2M) DCC 1.1 (0.2) N-HOSu dry DMF, 0?C→r.t., 12h

2) 2.0 (0.2M) biguanide DMF, r.t., 2 h

67%

Boc-HN COOBn

2) 1.1 (0.2) Fmoc-Cl 5 DIPEA, DMF, r.t., 12h

1) 0.25M TFA in CH2Cl2 0?C→r.t., 3h

10% Pd-C(wt/wt)DMF/1,4-cyclohexadiene (4:1)r.t., 3h

72%

s14 s15 s16

Scheme 3. Synthesis of the 2,4-diaminotriazine tagged aspartic acid monomer building block s17 used in the solid support synthesis of oligomer containing backbone type B (see Figure 1 of the manuscript).

HH

N

N N

H2N

NH2

Fmoc-HN COOBnH

N

N N

H2N

NH2

s17

Fmoc-HN COOHH

N

N N

H2N

NH2

82%

7

Cbz-HN

H2NOC

COOtBu1) 0.5 equiv. (0.25M) Lawessson's reagent dry THF, r.t., 12h 75% Cbz-HN

H2N(HN)C

COOtBu

1) 10% Pd-C(wt/wt) DMF/1,4-cyclohexadiene (4:1) r.t., 3h 72%

s18 s19 s20

Scheme 4. Synthesis of the 2,4-dioxotriazine tagged Asp-Asp dipeptide monomer building block s23 used in the solid support synthesis of oligomer containing backbone type B (see Figure 1 of the manuscript).

HHCbz-HN COOtBu

H

HN

HN N

O

O

s21

Fmoc-HN COOtBuH

HN

HN N

O

O

67%

2) 4 (1) MeI CH2Cl2, r.t., 12h3) 1 (7.5) aq. K2CO3, r.t., 10 min.

4) 1 (0.2) NH4Cl dry MeOH, reflux, 3h

92%

81%

1.0 (0.25) PhOOCNHCOOPh2 DBU, dry CH2Cl2, r.t., 2h

2) 1 (0.18M) FmocCl 1 (0.18M) DIPEA DMF, r.t., 12h 61%

1) 0.25M TFA in CH2Cl2, 0?C→r.t., 3h

2) 1 (0.18) NH2-Asp(OtBu)-OBn 1 (0.18) EDCI,1 (0.18) HOBt 1 (0.18) DIPEA, dry DMF, r.t., 8h

52%

s22

Fmoc-HNH

HN

HN N

O

O

NH

COOBn

OCOOtBu

s23

Fmoc-HNH

HN

HN N

O

O

NH

COOH

OCOOtBu

10% Pd-C(wt/wt)DMF/1,4-cyclohexadiene (4:1)5-10?C, 2h

70%

H H

HCl salt

8

Cbz-HN

HOOC

COOtBu

1) 10% Pd-C(wt/wt), H2 MeOH, r.t., 6h

s24 s25

Scheme 5. Synthesis of the 2-amino,4-oxotriazine tagged Asp-Asp dipeptide monomer building block s29 used in the solid support synthesis of oligomer containing backbone type B (see Figure 1 of the manuscript).

HCbz-HN COOtBu

H

N

N N

Cl3C

NH2

s27

Fmoc-HN COOtBuH

N

HN N

O

NH2

85%

1) 1 equiv. (0.2M) DCC 1 (0.2) N-Hosu dry THF, 0?C→r.t, 12h

2) 1 (0.18M) FmocCl 1 (0.18M) DIPEA DMF, r.t., 12h 85%

1) 0.25M TFA in CH2Cl2, 0?C→r.t., 3h

2) 1 (0.18) NH2-Asp(OtBu)-OBn 1 (0.18) EDCI,1 (0.18) HOBt 1 (0.18) DIPEA, dry DMF, r.t., 5h

54%

s28

Fmoc-HNH

N

HN N

O

NH2

NH

COOBn

OCOOtBu

s29

Fmoc-HNH

N

HN N

O

NH2

NH

COOH

OCOOtBu

10% Pd-C(wt/wt)DMF/1,4-cyclohexadiene (4:1)5-10?C, 2h

75%

2) 1 (0.15)

dry THF, r.t, 12h

NH

NH

NH

CCl3H2Ns26

Cbz-HN COOtBuH

N

HN N

O

NH2

1.2 (1) aq. NaOH, dioxaner.t., 2h

75%

H H

9

HN

NC

Boc

NC

HN

NH2

NHN N

NHN

H2N

NH2

Boc

N N

NNH2

H2N

NH2

KOH (cat.), 2-ethoxyethanol

120 oC, 2.5h

1) 4.5N HCl(MeOH) EtOAc, 1.5h, r.t., quant.

83%

2) Amberlite resin IRA-400 (OHŠ) 0.5h, r.t. 81%

COOBnO

OBnHN

ON

N N

NH2

H2N

H

61%

2 (0.37) NaBH3CNMeOH/AcOH3h, r.t.

NH2. HClEtOOC

COOt-BuBr2 (2) DIPEA, DMF, r.t.,

Ethyl glycine2) 1.2 (0.17) NaHCO3, 1.2 (0.17) Fmoc-Cl

1) 1.2 (0.17) LiOH 1,4-dioxane/H2O, 4 oC

N

OCOOtBu

FmocHO DMF, 16 h, r.t.

OBnN

N

ON

N N

NH2

H2N

OtBuOOC

Fmoc

H2, 10% Pd/CDMF

HON

N

O N

NN

NH2

NH2

OCOOtBu

Fmoc

+

N

OCOOtBu

FmocHO1.7 (0.28) EDC?HCl

80%

Scheme 6. Synthesis of the 2,4-diaminotriazine tagged dipeptiod monomer building block s35 used in the solid support synthesis of oligomer containing backbone type C (see Figure 1 of the manuscript).

2 equiv. (0.37M)

0.5 (0.5)

66%

60%

1 (0.16)

60%

s30 s31 s32

s33

S33

s34s35

10

NH2

+H3NO

Cl-

+

OBnBr

O

2 (1.7) DIPEANH2

HN

OO

BnO

1.1 (0.8) (Boc)2O1.1 DIPEA

NH2N

OO

BnO

Boc0.4 (0.1) Lawesson's reag., THF, r.t., 18h

NH2N

SO

BnO

Boc

r.t., 1h

PhO NH

OPh

O O

NH2+N

NH2O

BnO

Boc

-OOCCF3

1) 10 (2.8) MeI, CH2Cl2, r.t., 3h

DMF, r.t., 2hTHF/H2O, r.t., 18h 82%

1.9 (0.4) DBU, CH2Cl2, r.t., 2 h

2) 1 (2.2) Na2CO3, 10 min.

Cl-TFA/CH2Cl2 (1:1)

OBnBoc-N

ON

HN NH

O

O

OBn+H2NON

HN NH

O

O

1) 1.3 (0,23) FmocCl 2.7 (0.48) DIPEA

NON

HN NH

O

O

OHFmoc

BnOOCHN COOtBu

BnOOC Br+

NH2.AcOHBuOtOC

3.5 (0.8) DIPEA, DMF

NN

O

OBnO

COOtBu

N NH

NH

O

O

Fmoc

100%

NN

O

OHO

COOtBu

N NH

NH

O

O

Fmoc

BnOOCHN COOtBu

0.5 equiv. (0.5M)

41% 93%

3) 1 (0.37) NH4Cl, CH3OH 60oC, 3h

95%

1 (0.2)

65%88%63%

2) DMF/C6H8 (6:1) Pd(black)

1.3 (0.18) EDC.HCl, DMF

1.3 (0.18)

61%

DMF/C6H8 (5:1)Pd (black)

64%

1 (0.26)

1.8 (0.43)77%

Scheme 7. Synthesis of the 2,4-dioxotriazine tagged dipeptiod monomer building block s44 used in the solid support synthesis of oligomer containing backbone type C (see Figure 1 of the manuscript).

s36 s37 s38

s39s40s41s42

s43 s44

r.t., 16hr.t., 2h

monitored by TLC

11

H2N

H2N

O

CO2H

H2N

Boc-HN

O

CO2HBoc-HN

H2N

CO2HBoc-HN

CBz-HN

CO2H

Boc-HN

CBz-HN

NN

N

H2N

NH2

Boc-HN

H2N

NN

N

H2N

NH2Boc-HN

CBz-N

NN

N

H2N

NH2

OEtO

Boc-HN

CBz-N

NN

N

H2N

NH2

OHO

Boc-HN

CBz-N

NN

N

H2N

NH2

HN

MBHA-resin

1.5 equiv. (0.4M) Boc2O10% aq. K2CO3

1,4-dioxane, r.t., 16h

1.1 (0.2) PhI(OAc)2MeCN/AcOEt/H2O (2:2:1)

r.t., 16 h

81% 60%

1.5 (0.3) CbzCl10% aq. Na2CO3

1,4-dioxane, r.t., 16h

1) 1 (0.03) DCC, 1 (0.03) HOBt DMF, r.t, 16h2) 4 (0.04) Biguanide.HCl, r.t., 5d

40%

85%

(wt/wt) 10% Pd/C, H2MeOH, r.t., 12h

quant.

1) 1 (0.07)

1% ACOH in MeOH r.t., 2h

OOEtH

O

2) 1.2 (0.08) NaBH3CN r.t., 16h

3) 2 (0.07) CbzCl AcOEt/H2O 0→r.t., 16h

74%

3 (0.3) aq. LiOHTHF, r.t., 6h

82%

1) 1 (0.1) HATU, 2 (0,2) DIPEA MBHA resin[2], DMF, r.t, 16h2) Ac2O/NMP/pyridine (1:2:2) r.t, 45 min.

loading capacity = 60 µmol/g(by Kaiser test)[3]

Scheme 8. Synthesis of the 2,4-diaminotriazine tagged dipeptiod monomer building block s52 used in the solid support synthesis of oligomer containing backbone type D (see Figure 1 of the manuscript).

s45 s46s47 s48

s49s50s51s52

s53

O

12

Experimental details for the preparation of s6 and s13 (as representative examples):

Experiments for Scheme 1: (S)-Benzyl 2-(tert-butoxycarbonylamino)-4-(4,6-diamino-

1,3,5-triazin-2-yl)butanoate (s2): To a stirred soln. of s1 (5.06 g, 15 mmol, Novabiochem) in 30

ml of DMF at 0°C, a soln. of HOBt (2.23 g, 16.5 mmol) in 50 ml of DMF and a soln. of DCC

(3.40 g, 16.5 mmol) in 20 ml of DMF were added under nitrogen atmosphere. The reaction

mixture was left at 0°C for 1h, and then at room temperature for 12h. The resulting mixture was

filtered through a sintered funnel, and the solids were washed with 20 ml of DMF. The washings

were combined with the filtrate and this soln. (containing the activated ester) was added slowly

to a stirred soln. of biguanide (3.03 g, 30 mmol) in 30 ml of DMF. The reaction mixture was

stirred at room temperature for 2h. The solvent was removed in vacuo and the residue was

purified by CC (SiO2; AcOEt/MeOH 100:0→99:1.0) to obtain 3.46 g (57.4%) of s2. TLC

(CH2Cl2/MeOH 9.5:0.5): Rf 0.3. 1H-NMR (300 MHz, (D6)DMSO): 7.27-7.43 (m, 6 arom. H,

NH(Boc)), 6.61 (s, 2 NH2), 5.12 (d, J = 15.9, PhCH2O), 4.0-4.1 (m, 1 H- C(α)), 2.37 (t, J = 7.8, 2

H-C(γ)), 2.02-2.16 (m, 1 H-C(β)), 1.83-1.97 (m, 1 H-C(β)), 1.37 (s, C(CH3)3). 13C-NMR (75

MHz, (D6)DMSO): 176.60 (C(2), C(4)), 172.35 (CO), 166.86 (C(6)), 155.54 (CO(Boc)), 135.94

(arom. C), 128.33 (arom. C), 127.92 (arom. C), 127.66 (arom. C), 78.16 (C(CH3)3), 65.74

(PhCH2O), 53.53 (C(α)), 34.25 (C(γ)), 28.11 (C(CH3)3), 28.04 (C(CH3)3), 28.02 (C(CH3)3),

27.76 (C(β)). ESI-MS (pos.): 425 (21.0, [M + Na]+), 403 (66.7, [M + H]+), 347 (100, [M -

(CH2=C(CH3)2)]+).

(S)-Benzyl 2-[(S)-2-{((9H-fluoren-9-yl)methoxy)carbonylamino}-4-tert-butoxy-4-

oxobutanamido]-4-(4,6-diamino-1,3,5-triazin-2-yl)butanoate (s5): To a suspension of s2 (0.6 g,

1.49 mmol) in 3 ml of CH2Cl2 at 0°C was added TFA (3 ml), and the reaction mixture was stirred

at room temperature for 2h. The solvent and TFA were evaporated in vacuo, the residue co-

evaporated with toluene (3 x 10 ml), and dried under hv for 2h to give the crude tBoc-

deprotected amine TFA salt which was used without further purification.

To a stirred soln. of (S)-Fmoc-Asp(OtBu)-OH (0.613 g, 1.49 mmol, Novabiochem) in 5

ml of DMF:CH2Cl2 (1:1) at 0°C, HOBt (0.242 g, 1.79 mmol) and EDCI (0.343 g, 1.79 mmol)

were added under nitrogen atmosphere. After 15 min, a soln. of the crude tBoc-deprotected

amine TFA salt in 6 ml of DMF:CH2Cl2 (1:1) was added, followed by the addition of DIPEA

(0.25 ml, 1.49 mmol). The reaction mixture was stirred at room temperature for 5h, evaporated in

vacuo and the residue was purified on CC (SiO2; hexane/AcOEt 100:0→30:70 with 1% Et3N) to

13

obtain 0.91 g (87.7%) of s5. TLC (AcOEt): Rf 0.7. 1H-NMR (300 MHz, CDCl3): 8.71 (d, J =

6.0, NH), 7.73 (d, J = 7.5, 2 arom. H), 7.54 (dd appearing as t, J = 7.5, 2 arom. H), 7.22-7.42 (m,

9 arom. H), 6.44 (d, J = 8.4, NH(Fmoc)), 5.69 (s, 2 NH2), 5.13 (d, J = 24.6, PhCH2O), 4.63 (dd, J

= 6.6, 14.4, H-C(α-Asp)), 4.54 (dd, J = 6.6, 12.0, H-C(α-Glu)), 4.46 (d, J = 6.9, H2CC(Fmoc)),

4.18 (dd appearing as t, J = 7.2, HC(Fmoc)), 2.82 (dd, J = 6.0, 16.8, 1 H-C(β-Asp)), 2.50-2.70

(m, 3H, 1 H-C(β-Asp), 2 H-C(γ-Glu)) 2.10-2.30 (m, H2C(β-Glu)), 1.42 (s, C(CH3)3). 13C-NMR

(75 MHz, CDCl3): 178.02 (C(2), C(4)), 171.21 (CO), 170.79 (CO), 170.67 (CO), 166.79 (C(6)),

155.92 (CO(Fmoc)), 143.72 (arom. C), 143.61 (arom. C), 141.21 (arom. C), 135.41 (arom. C),

128.53 (arom. C), 128.29 (arom. C), 128.12 (arom. C), 127.69 (arom. C), 127.04 (arom. C),

125.02 (arom. C), 119.95 (arom. C), 81.84 (C(CH3)3), 67.21 (CH2(Fmoc)), 66.99 (PhCH2O),

53.07 (C(α-Glu)), 51.42 (C(α-Asp)), 47.00 (CH(Fmoc)), 38.23 (C(β-Asp)), 34.12 (C(γ-Glu)),

27.96 (C(CH3)3), 27.06 (C(β-Glu)). ESI-MS (pos.): 718 (8.2, [M + Na]+), 696 (100, [M + H]+).

(S)-2-[(S)-2-{((9H-fluoren-9-yl)methoxy)carbonylamino}-4-tert-butoxy-4-

oxobutanamido]-4-(4,6-diamino-1,3,5-triazin-2-yl)butanoic acid (s6): To a stirred suspension of

10% Pd-C (1.3 g) in 10 ml of DMF was added a soln. of s5 (1.3 g, 1.87 mmol) in 22.5 ml of

DMF and 6.5 ml of 1,4-cyclochexadiene, and stirred at room temperature for 2h. The reaction

mixture was filtered through celite and concentrated in vacuo. The resulting residue was purified

by reverse phase CC (C18-silica gel; H2O/MeOH 100:0→20:80) to obtain 0.75 g (66.3%) of s6.

TLC (AcOEt/MeOH/H2O/CH3COOH 8:1.4:0.6:0.2): Rf 0.5. 1H-NMR (600 MHz, (D6)DMSO)):

12.68 (s, HOOC), 8.21 (d, J = 7.2, NH), 7.88 (d, J = 7.2, 2 arom. H), 7.70 (dd appearing as t, J =

9.0, 2 arom. H), 7.64 (d, J = 8.4, H-N(Fmoc)), 7.41 (dd appearing as t, J = 7.8, 2 arom. H), 7.31

(dd appearing as t, J = 7.8, 2 arom. H), 6.58 (s, 2 NH2), 4.41-4.46 (m, H-(Cα-Asp)), 4.18-4.31

(m, 4H, H-(Cα-Glu), H-C(Fmoc), H2CC(Fmoc)), 2.65 (dd, J = 4.2, 16.2, 1 H-C(β-Asp)), 2.46

(dd, J = 9.6, 16.2, 1 H-C(β-Asp)), 2.34-2.42 (m, H2C(γ-Glu)), 2.06-2.13 (m, 1 H-C(β-Glu)), 1.89-

1.97 (m, 1 H-C(β-Glu)), 1.37 (s, C(CH3)3). 13C-NMR (150 MHz, (D6)DMSO)): 176.63 (C(2),

C(4)), 173.10 (CO), 170.65 (CO), 169.12 (CO), 166.82 (C(6)), 155.64 (CO(Fmoc)), 143.72

(arom. C), 143.61 (arom. C), 140.58 (arom. C), 127.52 (arom. C), 126.95 (arom. C), 125.14

(arom. C), 119.98 (arom. C), 80.02 (C(CH3)3), 65.62 (CH2(Fmoc)), 51.86 (C(α-Glu)), 51.13

(C(α-Asp)), 46.49 (CH(Fmoc)), 37.53 (C(β-Asp)), 34.06 (C(γ-Glu)), 28.46 (C(β-Glu)), 27.59

(C(CH3)3). ESI-MS: 606 (100, [M + H]+).

14

Experiments for Scheme 2: (S)-Benzyl 5-amino-2-(tert-butoxycarbonylamino)-5-

oxopentanoate (s8):[4] To a solution of s7 (492.6 mg, 1.0 mmol, Novabiochem) in 10 ml of dry

DMF was added NaHCO3 (336 mg, 2.0 mmol) and benzyl bromide (1.2 ml, 5.0 mmol), and the

reaction mixture was stirred at room temperature for 48h. DMF was evaporated in vacuo and the

resulting residue was dissolved in AcOEt (50 ml). The org. soln. was washed with H2O (2 x 50

ml), 25 ml of sat. aq. NaHCO3 and 25 ml sat. aq. NaCl. The org. layer was dried (Na2SO4) and

evaporated in vacuo. The residue was purified by CC (SiO2; hexane/AcOEt 50:50→0:100) to

obtain 0.55 g (80.5%) of s8. TLC (AcOEt/MeOH/AcOH 9.5:0.5:0.1): Rf 0.9. 1H-NMR (300

MHz, (D6)DMSO): 7.23-7.34 (m, 5 arom. H), 5.98 (br. s, NH(CO)), 5.55 (br. s, NH(CO)), 5.25

(d, J = 7.5, NH(Boc)), 5.11 (d, J = 17.7, PhCH2O), 4.23-4.35 (m, H-C(α)), 2.05-2.32 (m, 2 H-

C(γ), 1 H-C(β)), 1.79-1.94 (m, 1 H-C(β)), 1.37 (s, C(CH3)3). FAB-MS (pos.): 359 (41.5, [M +

Na]+), 337 (8.0, [M + H]+), 2.37 (58.5, [M + H - Boc]+), 91 (100, [PhCH2]+).

(S)-Benzyl 2-(tert-butoxycarbonylamino)-5-imino-5-(methylthio)pentanoate (s9): To a

suspension of Lawesson’s reagent (0.607 g, 1.5 mmol) in 6 ml of THF was added a soln. of s8 in

THF (6 ml) under nitrogen atmosphere at room temperature and the reaction mixture was stirred

at room temperature for 12h. The reaction mixture was evaporated in vacuo and 50 ml of AcOEt

was added. The org. layer was washed with 25 ml of H2O, 25 ml of sat. aq. NaHCO3, dried

(Na2SO4) and evaporated in vacuo. The residue was purified by CC (SiO2; hexane/AcOEt

100:0→60:40) to obtain 0.815 g (77.0%) of thioamide intermediate. TLC (AcOEt): Rf 0.9. 1H-

NMR (300 MHz, (D6)DMSO): 8.30 (br. s, NH), 7.43 (br. s, NH), 7.20-7.33 (m, 5 arom. H), 5.25

(d, J = 8.4, NH(Boc)), 5.11 (s, PhCH2O), 4.18-4.32 (m, H-C(α)), 2.60-2.76 (m, H2C(γ)), 2.21-

2.34 (m, 1 H-C(β)), 1.80-1.95 (m, 1 H-C(β)), 1.35 (s, C(CH3)3). 13C-NMR (75 MHz,

(D6)DMSO): 208.81 (CS), 171.84 (CO), 156.25 (CO(Boc)), 134.92 (arom. C), 128.61 (arom. C),

128.54 (arom. C), 128.36 (arom. C), 80.69 (C(CH3)3), 67.45 (PhCH2O), 52.38 (C(α)), 40.87

(C(γ)), 33.23 (C(β)), 28.17 (CH3(Boc)). FAB-MS (pos.): 375 (100, [M + Na]+), 297 (72.5, [M-

(CH2=C(CH3)2)]+). 91 (58.0, [PhCH2]+).

To a stirred soln. of thioamide intermediate (0.42 g, 1.19 mmol) in 4 ml of dry CH2Cl2

was added CH3I (0.74 ml, 11.9 mmol) under nitrogen atmosphere at room temperature and the

reaction mixture was stirred at room temperature for 12h. To the reaction mixture, a soln. of

K2CO3 (0.164 g, 1.19 mmol) in 0.35 ml H2O was added and stirred for 10 min. The org. layer

15

was washed with H2O (2 x 10 ml), dried (Na2SO4) and evaporated in vacuo to give 0.43 g

(98.4%) of s9 which was used without further purification. TLC (hexane/AcOEt 1:1): Rf 0.45. 1H-NMR (300 MHz, (D6)DMSO): 9.04 (s, NH), 7.30-7.40 (m, 5 arom. H), 5.25 (br. s, NH(Boc)),

5.17 (d, J = 13.2, PhCH2O), 4.34-4.42 (m, 1 H-C(α)), 2.40-2.55 (m, 2 H-C(γ)), 2.15-2.31 (m, 4H,

1 H-C(β), H3C-S), 1.96-2.10 (m, 1 H-C(β)), 1.43 (s, C(CH3)3). 13C-NMR (75 MHz, DMSO-d6):

172.08 (CO), 155.33 (CO(Boc)), 135.24 (arom. C), 128.71 (arom. C), 128.70 (arom. C), 128.56

(arom. C), 128.39 (arom. C), 128.26 (arom. C), 79.89 (C(CH3)3), 67.11 (PhCH2O), 53.09 (C(α)),

36.09 (C(γ)), 29.88 (C(β)), 28.25 (C(CH3)3), 12.30 (SCH3). FAB-MS (pos.): 389 (4.4, [M +

Na]+), 367 (27.0, [M + H]+).

(S)-Benzyl 5-amino-2-(tert-butoxycarbonylamino)-5-iminopentanoate hydrochloride

(s10): To a stirred solution of s9 (0.42 g, 1.146 mmol) in 5 ml of dry MeOH, solid NH4Cl (0.061

g, 1.146 mmol) was added, and the reaction mixture was refluxed for 3h. The mixture was

concentrated by evaporation in vacuo to afford a residue, which was dissolved in a minimum

amount of MeOH. This methanolic solun. was added drop wise to 200 ml of ether resulting in

precipitation; the precipitated solid was filtered through sintered funnel and re-dissolved in 10 ml

of CH2Cl2; the resulting soln. was filtered through cotton and concentrated in vacuo and the

residue was dried under h.v. to give 0.365 g (85.8%) of s10 which was use without further

purification. 1H-NMR (300 MHz, (D6)DMSO): 9.03 (s, 3 NH), 7.26-7.41 (m, 5 arom. H), 5.79

(d, J = 7.5, NH), 5.17 (d, J = 23.0, PhCH2O), 4.34-4.42 (m, 1 H-C(α)), 2.82-2.95 (br. m, 1 H-

C(γ)), 2.35-2.50 (br. m, 1 H-C(γ)), 2.15-2.32 (br. m, 1 H-C(β)), 1.88-2.07 (br. m, 1 H-C(β)), 1.40

(s, C(CH3)3). FAB-MS (pos.): 336 (100, [M + H - HCl]+).

(S)-Benzyl 2-(tert-butoxycarbonylamino)-4-(4,6-dioxo-1,4,5,6-tetrahydro-1,3,5-triazin-2-

yl)butanoate (s11): To a stirred solution of s10 (0.20 g, 0.54 mmol) and

diphenylimidodicarbamate (0.277 g, 1.08 mmol) in 2.69 ml of CH2Cl2 was added DBU (0.16 ml,

1.08 mmol) and the reaction mixture was stirred at room temperature for 2h. Then, AcOH (0.10

ml, 1.62 mmol) was added and the mixture was evaporated in vacuo. The resulting residue was

purified by CC (SiO2; CH2Cl2/MeOH 100:0→98.5:1.5) with 1% AcOH to obtain 0.135 g

(61.9%) of s11. TLC (CH2Cl2/MeOH/AcOH 9.5:0.5:0.1): Rf 0.6. 1H-NMR (600 MHz,

(D6)DMSO): 12.01 (s, NH (base)), 11.22 (s, NH (base)), 7.30-7.41 (m, 5 arom. H, NH(Boc)),

5.11 (d, J = 22.8, PhCH2O), 4.05-4.10 (m 1H-C(α)), 2.52 (t, J = 7.8, 2H-C(γ)), 2.08-2.15 (m, 1H-

C(β)), 1.85-1.93 (m, 1H-C(β)). 1.37 (s, C(CH3)3). 13C-NMR (150 MHz, (D6)DMSO): 171.96

16

(CO), 168.66 (CO), 155.48 (CO(Boc)), 135.83 (arom. C), 128.32 (arom. C), 127.96 (arom. C),

127.71 (arom. C), 78.32 (C(CH3)3), 65.89 (PhCH2O), 52.79 (C(α)), 30.14 (C(γ)), 28.09

(C(CH3)3), 26.10 (C(β)). ESI-MS (pos.): 427 (71.4, [M + Na]+), 405 (35.7, [M + H]+), 349 (42.9,

[M - (CH2=C(CH3)2)]+), 305 (100, [M + H - Boc]+).

(S)-Benzyl 2-[(S)-2-{((9H-fluoren-9-yl)methoxy)carbonylamino}-4-tert-butoxy-4-

oxobutanamido]-4-(4,6-dioxo-1,4,5,6-tetrahydro-1,3,5-triazin-2-yl)butanoate (s12): To a

suspension of s11 (0.6 g, 1.48 mmol) in 3 ml CH2Cl2 at 0°C was added 3 ml of TFA and the

reaction mixture was stirred at room temperature for 2h and evaporated in vacuo. The resulting

residue was co-evaporated with toluene (3 x 10 ml) and dried under h.v. for 2h to afford the

crude tBoc deprotected amine TFA salt which was used without further purification.

To a stirred solution of (S)-Fmoc-Asp(OtBu)-OH (0.610 g, 1.48 mmol, Novabiochem) in

6 ml of DMF:CH2Cl2 (1:1), HOBt (0.24 g, 1.78 mmol) and EDCI (0.341 g, 1.78 mmol) were

added at 0°C under nitrogen atmosphere. After 15 min. a soln. of the crude tBoc deprotected

amine TFA salt in 6 ml of DMF:CH2Cl2 (1:1) was added, followed by the addition of 0.25 ml of

DIPEA (1.48 mmol). The reaction mixture was stirred at room temperature, for 5h and

concentrated in vacuo. The resulting residue was purified by CC (SiO2; hexane/AcOEt

100:0→40:60 with 1% AcOH) to obtain 0.86 g (83.2%) of s12. TLC (AcOEt): Rf 0.75. 1H-NMR

(600 MHz, (D6)DMSO): 12.01 (s, NH (base)), 11.21 (s, NH (base)), 8.43 (d, J = 7.8, NH), 7.88

(dd appearing as d, J = 7.8, 2 arom. H), 7.70 (dd appearing as t, J = 7.2, 2 arom. H), 7.62 (d, J =

8.4, NH(Fmoc)), 7.41 (dd appearing as t, J = 7.2, 2 arom. H), 7.27-7.38 (m, 7 arom. H), 5.11 (d, J

= 14.4, PhCH2O), 4.40-4.45 (m, H-C(α-Asp)), 4.32-4.37 (m H-C(α-Glu)), 4.26-4.33 (m,

HC(Fmoc)), 4.19-4.26 (m, H2CC(Fmoc)), 2.56 (dd, J = 4.2, 16.2, 1 H-C(β-Asp)), 2.48-2.54 (m,

H2C(γ)) mixing with signal from DMSO), 2.43 (dd, J = 10.2, 16.2, 1 H-C(β-Asp)), 2.12-2.19 (m,

1 H-C(β-Glu)), 1.92-2.01 (m, 1H-C(β-Glu)), 1.37 (s, C(CH3)3). 13C-NMR (150 MHz,

(D6)DMSO): 171.13 (CO), 171.05 (CO), 169.04 (CO), 168.54 (CO), 155.61 (CO(Fmoc)), 143.73

(arom. C), 143.61 (arom. C), 140.59 (arom. C), 135.64 (arom. C), 128.29 (arom. C), 127.94

(arom. C), 127.73 (arom. C), 127.51 (arom. C), 126.92 (arom. C), 125.17 (arom. C), 125.13

(arom. C), 119.98 (arom. C), 80.03 (C(CH3)3), 66.02 (CH2(Fmoc)), 65.64 (PhCH2O), 51.36

(C(α-Asp)), 51.09 (C(α-Glu)), 46.49 (CH(Fmoc)), 37.78 (C(β-Asp)), 29.94 (C(γ)), 27.58

17

(C(CH3)3), 26.13 (C(β-Glu)). ESI-MS (pos.): 720 (100, [M + Na]+), 698 (54.8, [M + H]+), 642

(32.2, [M - (CH2=C(CH3)2)]+).

(S)-2-[(S)-2-{((9H-fluoren-9-yl)methoxy)carbonylamino}-4-tert-butoxy-4-

oxobutanamido]-4-(4,6-dioxo-1,4,5,6-tetrahydro-1,3,5-triazin-2-yl)butanoic acid (s13): To a

suspension of 10%Pd-C (0.10 g) in 1 ml of MeOH was added a soln. of s12 (0.10 g, mmole) in

1.5 ml MeOH and 0.5 ml of 1,4-cyclohexadiene and stirred at 5-10°C for 6h. The mixture was

filtered through celite and the filtrate concentrated in vacuo. The resulting residue was purified

by reverse phase CC (C18-silica gel; H2O:MeOH 100:0→30:70) to give 0.070 g (80.5%) of s13.

TLC (AcOEt/MeOH/H2O/CH3COOH 8:1.4:0.6:0.2): Rf 0.55. 1H-NMR (600 MHz, (D6)DMSO):

12.65 (s, NH (base)), 12.10 (s, NH (base)), 11.20 (s, COOH), 8.18 (d, J = 7.2, NH), 7.88 (dd

appearing as d, J = 7.2, 2 arom. H), 7.70 (dd appearing as t, J = 8.4, 2 arom. H), 7.62 (d, J = 8.4,

NH(Fmoc)), 7.41 (dd appearing as t, J = 7.2, 2 arom. H), 7.31 (dd appearing as t, J = 7.8, 2 arom.

H), 4.39-4.45 (m H-C(α-Asp)), 4.19-4.39 (m, 4H, H-C(α-Glu)), H-C(Fmoc), H2-C(Fmoc)), 2.65

(dd, J = 4.2, 16.2, 1 H-C(β-Asp)), 2.52 (m, 2 H-C(γ)) mixing with DMSO), 2.46 (dd, J = 9.6,

16.2, 1 H-C(β-Asp)), 2.07-2.16 (m, 1 H-C(β-Glu)), 1.87-1.95 (m, 1 H-C(β-Glu)), 1.37 (s,

C(CH3)3). 13C-NMR (150 MHz, (D6)DMSO): 172.75 (CO), 170.75 (CO), 169.13 (CO), 168.73

(CO), 155.62 (CO(Fmoc)), 143.75 (arom. C), 143.60 (arom. C), 140.58 (arom. C), 127.51 (arom.

C), 126.94 (arom. C), 125.15 (arom. C), 119.99 (arom. C), 80.05 (C(CH3)3), 65.66 (CH2(Fmoc)),

51.20 (C(α-Glu)), 46.49 (CH(Fmoc)), 37.54 (C(β-Asp)), 30.15 (C(γ-Glu)), 27.59 (C(CH3)3),

26.61 (C(β-Glu)). ESI-MS (pos.): 652 (36.7, [M + 2Na]+), 630 (100, [M + Na]+), 608 (5.0, [M +

H]+).

18

Sequences synthesized for this study:

Table 1. HPLC and MS Data of Triazine Tagged A–D Oligomer Sequences.[a]

1 HOOCAspGlu(TNN)6 A 0→30%B in 30, 12.90 1873 1873 2 HOOCAspGlu(TNN)12 A 0→100%B in 30, 11.35 3730 3727 3 AcNHAspGlu(TNN)16 A 0→100%B in 30, 12.42 5007 5006 4 AcNHAspGlu(TOO)12 A 0→100%B in 30, 17.42[d] 3838(2Na+) 3793(2Na+) 5 AcNHAspGlu(TOO)16 A 0→100%B in 30, 15.92[d] 5038 5037 6 AcNHAspGlu[(TNN)(TOO)]6 A 0→100%B in 30, 15.21 3782 3779 7 HOOCAspGlu(TNO)12 A 0→100%B in 30, 30.35 3738 3738 8 HOOCAspAsp(TNN)6 A 0→20%B in 30, 14.21 1789 1789 9 HOOCAspAsp(TNN)12 A 0→100%B in 30, 11.43 3559 3559 10 HOOCAspAsp(TOO)4 A 0→30%B in 30, 18.39 1206 1206 11 HOOCAspAsp(TNO)6 A 0→70%B in 30, 17.67 1816(Na+) 1816(Na+) 12 HOOCAspAsp(TNO)10 A 0→100%B in 30, 14.40 3005(Na+) 3001(Na+) 13 NH2 IDAbackbone(TNN)8 A 0→100%B in 30, 9.6 2379 2379 14 NH2 IDAbackbone (

TNN)12 A 0→100%B in 30, 11.5 3559 3559 15 NH2 IDAbackbone (TOO)6 A 0→30%B in 30, 25.0 1782 1800 16 NH2 IDAbackbone (TOO)7 A 0→30%B in 30, 27.0 2080 2096 17 NH2 EDAbackbone (

TNN)12 B 0→30%B in 30, 11.96[e] 2549 (Na+) 2549 (Na+) [a] All sequences refer to triazine tagged oligomeric backbone sequences depicted in Figure 1. TN,N = 2,4-diaminotriazinyl; TO,O = 2,4-dioxotrazinyl; TN,O = 2-

amino-4-oxotriazinyl; for abbreviations please refer to footnote of Table 1 in main paper; AcNH = end NH2 group was acetylated after final FMOC-deprotection.

[b] Method A:. TFA/m-cresol (95:5); Method B: TFMSA/TFA/m-cresol (1:10:1). [c] MONO-Q HR 5/5 Pharmacia, 10 Ï 0.5 cm or Nucleogen-DEAE 60-7

Machery Nagel, 125 Ï 4, flow 1 ml / min. Mobile phase: eluant A: 10 mM Na2HPO4, H2O, pH 11.5; eluant B: 10 mM Na2HPO4, 1 M NaCl, H2O, pH 11.5. [d]

Nucleogen-DEAE 60-7 Machery Nagel, 125 Ï 4, flow 1 ml / min. Mobile phase: eluant A: 10 mM Na2HPO4, H2O, pH 7.0; eluant B: 10 mM Na2HPO4, 1 M NaCl,

H2O, pH 7.0. [e] Aquapore ODS 20 micron Brownlee, 250 Ï 10.0 mm, flow 4 ml / min. Mobile phase: eluant A: 0.1% TFA in H2O; eluant B: 0.1% TFA in

MeCN. [f] Matrix assisted laser-desorption ionization time-of-flight mass spectroscopy; matrix: 3-hydropicolinic acid or α-cyanohdroxycinnamic acid or 2,4,6-

trihydroxyacetophenone and ammonium citrate buffer.

No. Sequences[a] Deprotection[b] Methods

Analytical HPLC [c] ––––––––––––––––––––– gradient, tR [min.]

MALDI-TOF-M[f] [M + H]+ [M +H]+ (obs.) (calc.)

19

Studies referring to the instability of the Oligomer system D(TNN):

A 0.1% aq. TFA solution of D(TNN)10 at room temperature was found to decompose (12% remaining with 88% decomposition in 24 h

as judged by reverse phase HPLC traces and confirmed by MALDI-TOF measurements).

0hr: 1605736 100%2hr: 1416938 88%6 hr: 567207 35%24hr: 193344 12%

Rev. Phase HPLC traces of D(TNN)10 in 0.1% aq TFA solution (details of HPLC conditions in caption of Table 2).

MALDI-TOF MS of D(TNN)10 in 0.1% aq TFA, after 24hrs.

A much lesser degree of decomposition of D(TNN)12 is suspected under the UV-Tm measurement conditions at pH 7; for e.g. in

the case of the duplex formed by D(TNN)12 + poly-(dT), its UV-Tm value, after four heating-cooling-heating (0–90 °C) cycles, was

found to decrease from 47.4 to 43.4 °C which was accompanied by an overall decrease in hyperchromicity from 25 to 20%. A model

study on the stability of the dimer, D(TNN)2 •TFA salt in D2O by 1H-NMR-spectral analysis showed the appearance of glycinamide,

which increases with a concomitant loss of the methylene signal from the NH2COCH2NH– moiety of the parent dimer over a period

of 12 days. This indicated the decomposition of the dimer presumably via a β-elimination of the ammonium residue of the

NH2COCH2NH– moiety at the α-position of the triazine ring.

20

Preparation of tert-butyl (4,6-dioxo-1,3,5-triazin-2-yl)methylcarbamate (s56):

Boc-HNN

HOOCSH

NH-Ac

NH3, MeOH, 60oC, quant.

PhO NH

OPh

O O

DBU, CH2Cl2, r.t., 51%s54 Boc-HN

N

NHHN

O

OBoc-HN

NH2+

NH2

-OOCSH

NH-Ac

s55s56

A stirred solution of s54 (2.03 g, 13.0 mmol) and N-acetyl cytstein (2.12 g, 13.0 mmol) in

dry MeOH (20.0 ml) at 60oC, was gently bubbled with NH3 for 7 hours (MeOH was added to the

solution as needed). The reaction was then bubbled with N2 for 20 minutes and concentrated in

vacuo to afford 4.37 g of amidine s55 (100% yield) as a amorphous white solid. The crude solid

was used in the next step without further purification.

To a stirred solution of diphenyl imido dicarbonate (3.19 g, 12.43 mmol) in dry CH2Cl2

(40.0 ml) at room temperature, s55 (3.8 g, 11.3 mmol) was added. To this suspension DBU (3.38

ml, 22.6 mmol) was added and the clear yellow solution was stirred for 1 h at room temperature

The reaction mixture was then quenched with AcOH (1.29 ml, 22.6 mmol), adsorbed on silica

gel and purified by CC (silica gel; AcOEt/acetic acid 99:1→AcOEt/MeOH/acetic acid 84:15:1)

to afford 1.6 g (51% yield, for two steps) of s56 as a white amorphous solid. TLC

(AcOEt/MeOH/AcOH 90/10/1): Rf 0.70. 1H-NMR (600 MHz, DMSO-d6): 12.10 (br. s, 1H, N-

H), 11.20 (br. s, 1H, N-H), 7.10 (t, J = 5.5 Hz, NH-Boc), 3.88 (d, J = 5.5 Hz, N-CH2-), 1.37 (s,

9H, -C(CH3)3). 13C-NMR (75.9 MHz, DMSO-d6): 167.7, 155.8, 153.5, 78.5, 42.2, 40.3, 28.2;

ESI-MS (neg.): 241 (100%, [M - H]-).

21

pKa determination of the 2,4-dioxo-triazine heterocycle in tert-butyl (4,6-dioxo-1,3,5-triazin-2-

yl)methylcarbamate (s56):

Wavelenght (nm)

Absorbance

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

220 240 260 280 300 320

pH = 9.0

pH = 3.0

250.0 nm

232.5 nm

pH

Absorbance

0.1

0.15

0.2

0.25

0.3

0.35

0.4

2 3 4 5 6 7 8 9 10

(a) Effect of pH on the UV absorption spectrum of s56. (b) pKa determination of s56. pKa ˜ 6.0

s56 was dissolved in two batches of 400 ml phosphate buffer at pH = 6 (1.25 mM Na2HPO4, 12.5

µM Na2(EDTA)) to an approximate concentration of 40 µM. These solutions were titrated with

1.0M soln. of NaOH (till pH = 9.0) and 1.0M soln. of HCl (till pH = 3.0), respectively. Dilution

by the added base and acid was less than 0.5% and no correction was applied. The added

amounts of NaOH and HCl solutions (to increase or decrease of 0.5 point in the pH) were in the

µL range (< 50 µL) for measurement.

pKa determination of heterocycle 2,6-diaminopurine in 2,6-Diamino-9-(β-D-

ribofuranosyl)purine (following the same procedure as above):

Absorbance

0

0.2

0.4

0.6

0.8

1

1.2

220 240 260 280 300 320

Wavelength (nm)

pH 2.5

pH 6.2

0.05

0.1

0.15

0.2

0.25

0.3

0.35

2.5 3 3.5 4 4.5 5 5.5 6 6.5

Absorbance

pH

300 nm

(a) Effect of pH on the UV absorption spectrum. (b) pKa determination. pKa ˜ 4.4

Boc-HN

N

NHHN

O

O

s56

OHO

HO

NN

N N

NH2

NH2

22Hydrolytic instability of 2,4-dioxo-triazine containing sequences – instability of AspGlu(TOO)16.

0

5

10

15

0 20 40 60 80 100

of AspGlu(TOO)16

Comparison of UV-Tm curves from 1st and 2nd Tm determination experiments

10µM, 1M NaCl, 10 mM Na2HPO4, 0.1mM Na2EDTA, pH = 7

1st Tm measurement

2nd Tm measurement

h

h

c

% H ( 2 4 0 n m )

T (°C)

0.95

1

1.05

1.1

1.15

1.2

0 20 40 60 80 100

A b s ( 2 4 0 n m )

T (°C)

1st Tm measurement

2nd Tm measurement

h

c

c

h

24 hr.t.

Before Tm measurement

After 1st Tm measurement

After 2nd Tm measurement

of AspGlu(TOO)16

Comparison of HPLC curves from before, 1st and 2nd Tm determination experiments

10µM, 1M NaCl, 10 mM Na2HPO4, 0.1mM Na2EDTA, pH = 7

Ion exchange HPLC: MONO-Q column HR 16/10; 0→100%B in 30 min. Buffer A: 10mM Na2HPO4, pH ­ 11.3. Buffer B: 10mM Na2HPO4, 1M NaCl, pH ­ 11.3

23a) at pH 5.0 (b) at pH 7.0

2 0C, λmax 233 nm, abs 0.5171720 0C, λmax 234 nm, abs 0.50879

80 0C, λmax 237 nm, abs 0.4536960 0C, λmax 236 nm, abs 0.47533

40 0C, λmax 235.5 nm, abs 0.49210

0

0.2

0.4

0.6

0.8

1

220 240 260 280 300 320Wavelength (nm)

A b s o r b a n c e

Wavelength (nm)

A b s o r b a n c e 2 0C, λmax 238 nm, abs 0.8513520 0C, λmax 242 nm, abs 0.85458

79 0C, λmax 250 nm, abs 0.9385560 0C, λmax 248 nm, abs 0.93608

40 0C, λmax 246 nm, abs 0.89146

0

0.5

1

1.5

220 240 260 280 300 320

The temperature dependent UV spectra of AspGlu(TOO)12: c =10 µM, 1 M NaCl, 10 mM Na2HPO4.7H2O, 0.05 mM Na4EDTA.4H2O, 0.05 mM EDTA.

a) b)

A/270 nm

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 20 40 60 80 1000.25

0.3

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0 20 40 60 80 100

A/260 nm

% AspGlu(TNN)16 % IDAbackbone(TNN)12

AspGlu(TNN)16 + d(T16) IDAbackbone(TNN)12 + r(T12)

Job-plots showing the 1:1 ratio of the pairing partners in the duplex formed between a) AspGlu(TNN)16 + d(T16) and b) IDAbackbone(TNN)12 + r(T12). Mixing curves at 10 and 15 °C respectively in 5+5 µM in 1M NaCl, 10 mM aq. NaH2PO4, 0.1 mM Na2EDTA, pH 7.0 .

24

-30

-20

-10

0

10

20

30

40

50

220 240 260 280 300 320

λ/nm

AspGlu(TNN)16

0?C

60

CDθ/mdeg

d)

CD-spectrum of self-pairing of AspGlu(TNN)16, Tm = 22 °C (222 nm). AspGlu(TOO)16 alone shows no temperature dependence of CD-spectrum. Measurements were made with c ˜ 10 µM (1:1) in 1M NaCl, 10 mM aq. NaH2PO4, 0.1 mM Na2EDTA, pH 7.0. Temperature increments in 5°C steps.

25

References:

1) (a) T. Wagner, B. Han, G. Koch, R. Krishnamurthy, A. Eschenmoser, Helv. Chim. Acta

2005, 88, 1960. (b) B. Han, V. Rajwanshi, J. Nandy, R. Krishnamurthy, A. Eschenmoser,

Synlett, 2005, 744.

2) (a) L. Christensen, R. Fitzpatrick, B. Gildea, K. H. Petersen, H. F. Hansen, T. Koch, M.

Egholm, O. Buchardt, P. E. Nielsen, J. Coull, J. Peptide Sci. 1995, 3, 175. (b) T. Koch, H. F.

Hansen, P. Ansersen, T. Larsen, H. G. Batz, K. Otteson, H. Orum, J. Peptide Res. 1997, 49,

80.

3) V. K. Sarin, S. B. H. Kent, J. P. Tam, R. B. Merrifield, Anal. Biochem. 1981, 117, 147.

4) Prepared according to a procedure described for benzylation in the asparagine series: S.

Nakabayashi, C. D. Warren, R. W. Jeanloz, Carbohydrate Research, 1998, 174, 279.