האוניברסיטה העברית בירושלים הפקולטה לרפואה, בית הספר לרוקחות Novel Analogues of Gene Silencing Agents Based on Positively Charged PNA מועתז חג' יחיא ב

תמליל

1 האוניברסיטה העברית בירושלים הפקולטה לרפואה, בית הספר לרוקחות ovel Analogues of Gene Silencing Agents Based on Positively Charged PA מועתז חג' יחיא בהנחיית: פרופ' יהושע כצהנדלר במסגרת הלימודים לקבלת תואר מוסמך בביה"ס לרוקחות האוניברסיטה העברית בירושלים אפריל 2014 ניסן תשע"ד

2 Acknowledgments I would like to thank from the bottom of my heart to my supervisor, Prof. Joshua Kazhendler, for his endless help, support, understanding and patience.

3 תקציר שיטות להשתקת גנים נחקרו ממספר כיוונים שונים: א( אנלוגים שלDA ב( אנלוגים של sira ג( אנלוגים של.PA באשר ל-א( DA בעל מבנה הטעון שלילית שאינו חודר את ממברנת התא ובכדי להחדירו יש צורך בשימוש בנשאים כגון; פוליאמינים, פוליארגינינים, ליפופקטמינים או sira עובר דגרדציה מהירה בפלזמה.)CPP באשר ל ב( ה פפטידים להחדרה תאית ( ובציטופלזמה, בנוסף נדרש נשא עזר על מנת להחדירו לתא. ולכן בחירתנו הייתה פפטידים של חומצות הגרעין ( )PAs שהם אוליגומרים של בסיסים נוקלאיים שבהם השלד הוחלף בשלד המורכב מיחידות חוזרות של אמינואתיל גליצין.במלים אחרות ניתן להגדיר את ה- PA כ- DA PA יש המורכב משלד של פפטיד ניטראלי במקום שלד של סוכר-פוספאט שטעון שלילית.ל- יציבות כימית ועמידות בפני ביקוע ודגרדציה אנזימטית ולכן לא צפוי גם שיעבור דגרדציה בתוך התא למרות הובלתו הקשה בתוך התא.ה- PA מסוגל לזהות רצפים ספציפיים של DA ו- RA Watson-Crick כך שהקומפלקסים )דופלקס ותריפלקס( שנוצרים וליצור איתם קשרי מימן עפ"י PA יכולת היברידיזציה גבוהה ויציבות הם בעלי יציבות תרמית וחוזק יוני מוגברים. ל- להיות בעל שימוש נרחב אנזימטית וכימית משופרות ביחס לחומצות הגרעין, דבר הגורם ל- PA מסוגל לעכב שעתוק ותרגום גנטי PA בתחום התרפאותי והדיאגנוסטי. מחקרי עבר מראים ש- באותה מידה, ולכן זה הופך אותו לכלי חדש בתרפיה המבוססת על אנטי-גן ואנטי-סנס, ובנוסף DA למטרות מחקר בשל מאפייניו המיוחדים. אולם, מצד שני, החדרת ה- לשמש גם כתחליף PA אל תוך התא מהווה מגבלה כמו בשאר המולקולות בעלות מסות גבוהות. על מנת להתגבר על PA מכיוון שהם מגבירים את מגבלה זו יש לקשור צורונים בעלי מטען חיובי על פני רצף ה- מספר יתרונות פרמקולוגיים כגון: א( חדירותו לתא. ידוע מהספרות המדעית שלקבוצות קטיוניות שיפור הפרמטרים הפרמקוקינטיים, ב( הגברת חדירות תאית ו-ג( שיפור המעבר של מחסום דם- מוח. מטרתנו בעבודה זו הינה להכין אנלוגים חדשניים של חוסמי שעתוק גני )משתקי גנים( הבנויים על שלד של PA,אשר: 1.מכילים אלמנטים בעלי מטען חיובי לאורך רצף ה- PA והחדירות. שתפקידם להגביר את המסיסות 2. מכילים אלמנטים)שיירים של דיאמינופורין ואנטרקלאטורים( לחיזוק ההיברידיזציה והקישור עם הגדיל המשלים של. DA בנוגע ל 1 ; התמקדנו במהלך עבודתנו להכין אבני בניין שמבוססים על שלד של PA ונושאים איתם שרשראות צדדיות בעלות מטען חיובי )דימיתי לאמין וגואנידין(, ובנוסף נגזרות ליזין המעוצבות לעמוד בפני דגרדציה ע"י פרוטאזות למיניהם.

4 בנוגע ל 2; הרעיון הוא ל נצל את היתרון של מספר תצורות שתספק להיברידPA DA- ייצוב נוסף בשל קשרי מימן נוספים או אינטראקציות.π-stacking בהקשר זה הגישה שלנו היא להכין את: א. אבן בניין של PA שנושאת יחידת דיאמינופורין אשר נועדה להחליף את הבסיס אדנין על גדיל דיאמינופורין יוצר שלושה קשרי מימן עם תימין עמיתו במקום שניים עם אדנין. ב. אבני הבניין של.PA,PA הנושאות אלמנטי אינטרקלאציה כגון; אנטראקינון, פירן ואנטראצן שנועדו להגביר את הקישור דרך אינטראקציות π-stacking נוספות בין הבסיסים הנוקלאיים. מאז שכבר הוכח כי צורונים קטיוניים מגבירים מסיסות וחדירות של PA לתא ואנטרקלאטורים מצורפים עשויים להגביר את רמת הקישור בדופלקס, PA:DA כאן בעבודה זו הכנו שלד שלPA אשר: א. נושא יחידות קטיוניות }דימיתילאמין )6,20 ), גואנידין )22({, מתיל על שלד )7(. ב. אנטרקלאטורים: }אנטראקינון )5(, פירין )21({. בנוסף הכנו גם יחידות קטיוניות ואנטרקלאטורים שנשתלו על השייר של ליזין כ-: א. }דימיתיל ליזין )24( )-H) }(Fmoc-Lys(Me 2 ב. }אנטראצן )26(, פירין )21({. על מנת לשפר קישור בין T והבסיס המשלים שלו צירפנו דיאמינופורין )23( על שלד של PA על מנת ליצור שלושה קשרי מימן במקום שניים במקרה שלT-A. הרצפים הנידונים של PA הוחדרו לתוך תאים סרטניים ויכולתם לדכא את ביטוי הגני של - glypican 1 נבדקה. תוצאות הניסוי מראות ששני המבנים דיכאו ועיכבו באופן משמעותי את הביטוי של glypican באחוזים 40-50% והוכיחו את יעילותן של הקבוצות הנידונות.

5 ABSTRACT Gene silencing methods have been approached from several directions. a. DA analogues, b. sira analogues, c. PA analogues [1]. Regarding to a. DA is negatively charged therefore cannot permeate cell membrane and in order to penetrate it we need to use some carrier agents as polyamines, polyarginines, lepofectamines or cell penetrating peptides (CPP). Regarding to b. sira is rapidly degraded in plasma and in cellular cytoplasm, resulting in short half-life. Further sira molecules do not penetrate into the cell efficiency, necessitating the use of a carrier for proper delivery. For this purpose, we chose the peptide nucleic acids (PAs). These are nucleobase oligomers in which the entire backbone has been replaced by a backbone composed of -(2-aminoethyl) glycine units [2,3]. In other words, PA can be regarded as DA with a neutral peptide backbone instead of a negatively charged sugar phosphate backbone. It is chemically stable and resistant to hydrolytic (enzymatic) cleavage and thus not expected to be degraded inside a living cell, although its transportation within the cell is difficult. PA is capable of recognizing specific sequences of DA and RA obeying the Watson Crick hydrogen bonding scheme, and the hybrid complexes exhibit extraordinary thermal stability and unique ionic strength effects. It may also recognize duplex homopurine sequences of DA to which it binds by strand invasion, forming a stable PA/DA/PA triplex with a looped-out DA strand. PA exhibits superior hybridization characteristics and improved chemical and enzymatic stability relative to nucleic acids, which is why PA finds major applications in the diagnostic and pharmaceutical fields. Studies indicate that PA is capable of inhibiting transcription as well as translation, so it can be used as a new tool for antigene and antisense therapy. wing to its superior properties, PA could replace DA as a probe for many investigation purposes. PAs can be conveniently delivered into cells in complex with DA and cationic lipid. However, as with other high molecular mass drugs, the delivery of PA, involving passage through the cell membrane, appears to be a general problem. In order to overcome the penetration restrictions, positively charged moieties linked to the PA sequence seems to enhance their cell uptake. It is well documented in the literature that cationic residues confer several pharmacological advantages to many substances as:

6 a. improved pharmacokinetics parameters in circulation. b. enhanced cell permeation. c. increased BBB transport properties. The objective of our research is to prepare new types of gene silencing constructions based on the PA backbone: 1. Comprising of positively charged elements along the polymeric chain, aimed to increase the solubility and the cell permeability of PA analogues. 2. Comprising of binding anchors (diaminopurine and intercalatorical residues) aimed to enhance the hybridization to the complementary DA strand. Regarding to 1, in this context our concern was focused to prepare new building blocks based on PA back bone which bears a positively charged side chain (dimethyl amine and guanidine group). Also on modified lysine derivatives designed to avoid the proteases degradation. Regarding to 2, the idea is to take advantage of several formations that will provide the PA-DA hybrid either additional stabilization due to auxiliary hydrogen bonding or stacking interactions. In this regards our approach is to prepare: a. PA building block comprising of diaminopurine aimed to replacing the adenine base on the PA strand. Diaminopurine forms three hydrogen bonds with its counterpart thymine instead of two with adenine. b. PA building blocks including intercalating agents, the anthraquinone and pyrene components will increase binding through additional stacking interactions between the base pair. Since it has been shown that cationic moieties enhance cell uptake, PA solubility and attached intercalator agents may increase the binding level of the PA:DA duplex, here in, we have prepared PA backbone: a. bearing cationic units {guanidine (22), dimethylamine (6,20)},and -methylated backbone (7) and b. intercalators: {anthraquinone (5), pyrene (21)}.In addition we have also prepared cationic units and itercalators on lysine side chain as: a.dimethyl Lysine (Fmoc- Lys(Me 2 )-H) (24)} and b. {anthracene(26), pyrene (21)}.In order to enhance binding between T and his complementary base we have also attached diaminopurine

7 (23) to the PA backbone in order to form three hydrogen bonds instead of two in the case of T-A pairing. These PAs were transfected into tumor cells and their ability to suppress the glypican-1 transcripts evaluated. Both of the transfected structures suppressed significantly the expression of glypican-1(40-50%) and indicated the efficiency of the studied modifying agents.

8 Table of contents Introduction....1 Hypothesis & Research objectives..14 Experimental Results.. 32 Discussion Bibilography....55

9 List of figures and schemes Fig.1: Transcription inhibition by PA's..3 Fig.2: ovel types of polyamide building blocks Fig.3: PA structures based on cyclic scaffold...6 Fig.4: Cationic derived PA Fig.5: Hydrogen bonding between DAP/T ; G/C...11 Fig.6: Intercalator between PA-DA Duplex Fig.7: Interchalator connection modes on the PA backbone Fig.8: Intercalator Model A & Model B Fig.9: Monomers of cationic residue Fig.10: DAP monomer Fig.11: Monomers of interchalator residue Fig.12: PA sequences against glypican-1 mra: RV11 & RV Fig.13: The final PA sequences (RV11 & RV12) against glypican-1 mra...47 Fig.14: Fmoc-protected PA monomers Fig.15: The effect of RV11,RV12 &sira on glypican-1expression Scheme 1: PA backbone preparation (aeg) Scheme 2: Preparation of antraquinone derivative...36 Scheme 3: Linking anthraquinone-propionic acid & dimethylaminobutyric acid to aeg 37 Scheme 4: Methylation of the PA backbone...37 Scheme 5: PA backbone preparation....38

10 Scheme 6: Linking of dimethylamino butyric acid & pyrenebutyric acid to PA backbone...38 Scheme 7: Preparation of Di-BC-S-methylisothiourea Scheme 8: Preparation of primary amine on PA backbone Scheme 9: Attaching of Di-BC-S-methylisothiourea to PA backbone. 40 Scheme 10: Preparation of tetra-bc DAP-yl-acetic acid Scheme 11: Attaching tetra-bc DAP acetic acid to PA backbone Scheme 12: Cleavage of the benzyl protecting group under hydrogenation...42 Scheme 13: Preparation of dimethyl lysine {Fmoc-Lys (Me 2 )-H} Scheme 14: Preparation of pyrene & anthracene-lysine derivatives Scheme 15: General procedure for diethylenetriamine synthesis Scheme 16: Anthraquinone spacer synthesis- Res (28) Scheme 17: Anthracene spacer synthesis- Res (30-3) Scheme 18: Complete preparation route via method A Scheme 19: Complete preparation route via method B...50

11 Abbreviations AC: Acetonitrile AEAE: 2-Amino-Ethyl Aminoethanol Aeg: Amino Ethyl Glycine Aep: Amino Ethyl Pirrolidine Aepip: Amino Ethyl Pirrollidine AIDS: Acquired Immune Deficiency Syndrome Arg: Arginine B: ucleobase BAB: Bromoacetyl Bromide BEP: Backbone Extended Pirrolidine BP: (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate Bz: Benzyl C: Cytidine or Cytosine PA monomer o C: Degree Celsius CX: Cyclooxygenase DAP: 2,6 diamino Purine DBU: 1,8-Diazabicyclo[5.4.0]undec-7-ene DCC: Dicyclohexyl Carbodiimide DCM: Dichloromethane DDW: Double Distilled Water DIEA (DIPEA): Diisopropyl Ethyl Amine DMAP: Dimethylamino Pyridine

12 DMF: Dimethyl Formamide DMS: Dimethyl Sulphoxide s: itrobenzene solfunyl DA: Deoxyribonucleic Acid ESI-MS: Electron Spray Impact Mass Spectrometry. EtH: Ethanol Fmoc: Fluorenyl Methylene xy Carbonyl G: Guanine g: Guanosine or Guanine PA Monomer Gly: Glycine HBTU: -(Benzotriazol-1-yl)-,,, -tetramethyluronium hexafluorophosphate HBT: Hydroxy Benztriazole Lys: Lysine MR: uclear Magnetic Resonance MP: -Methyl Pyrrolidone SAIDs: on stereoidal anti-inflamatory drugs D: ligo Deoxyribonucleic Acid PamA: Polyamine ucleic Acid PA: Polyamide (peptide) ucleic Acid Pro: Proline RA: Ribonucleic Acid r.t.: Room Temperature SPS: Solid Phase Synthesis

13 t: Thymidine or Thymine PA monomer T: Thymine TBBA: Tert-Butyl Bromoacetate tbc (BC): Tert-Butyl xycarbonyl tbu: Tert-Butyl TEA: Triethyl Amine TES: Triethyl Silane TFA: Trifluoroacetic Acid Tm: Melting Temperature TMS: Trimethyl Silyl TMS-Im: Trimethyl Silyl Imidazol

14 Introduction 1

15 Scientific Background Synthetic oligonucleotides have been used to inhibit DA replication and protein synthesis with very high specificity 1. Antisense oligonucleotides and ribozymes were studied in many laboratories during the last decade 2-5 as possible therapeutic approaches against AIDS. Recent advances in antisense technology have been focused on modifying oligonucleotides in order to achieve improved nuclease resistance and increased binding affinity. These include: (a) Backbone modifications (b) Sugar modifications, and (c) Base modifications. The first generation of antisense oligonucleotides was based on chemical modifications in which the backbone phosphor-di-ester bond was replaced by (a) phosphorothioates 6, (b) phosphorodithioates 7 (c) methylphosphonates 8, and (d) phosphoramidates 9. The second generation of antisense D's comprise of complete phosphate backbone replacement. This includes carbonates 10, carbamates 11, urea 12, guanidine 13, amides 14, amines 15 and alkanes 16. An additional approach is related to the -anomeric analogues 30. An attractive approach in the development of antisense agents for DA and RA recognition is the polyamide (peptide) nucleic acid (PA). PAs are the first successful substitute for the sugar-phosphate backbone that have displayed equal or better biding affinity than natural DA or RA 18. In contrast to the various backbone units, PAs do not bear any structural resemblance to natural oligonucleotides. PAs bind to an oligonucleotide sequence either via a parallel mode, where the PA amino terminus is aligned with the 5 ' end of DA, or via an anti-parallel mode (aligned with the 3 ' end). Hybridization through the antiparallel mode was found to be significantly more stable than the corresponding parallel hybrid and imparts an extra Tm stability of C/modification and C /modification for PA-RA and PA-DA duplexes, respectively. The alternative parallel binding mode is still as stable as DA-RA or DA- DA duplexes. Transcription inhibition by PA's (figure 1) can occur either by triple helix formation or by strand invasion in which the PA displaces one DA strand in the DA duplex to form a PA-DA hybrid (strand displacement). Such an event may then be followed by binding of an additional PA oligomer, generating a local (PA) 2 -DA 2

16 triple helix. Both PA strands must be oriented either parallel or antiparallel to the DA strand. Strand invasion is common with homopurine - homopyridine duplex. Figure 1: Transcription inhibition by PA's Two pivotal obstacles are implicated with the application of PA systems: i) Low solubility, and ii) Poor cellular uptake. In this context, in order to cope with these hurdles, modified polyamide building blocks were introduced as depicted in figure 2: 3

17 Figure 2: ovel types of polyamide building blocks Compound A represents the original structure (ielsen) of a PA (aeg) unit where the backbone is comprised of a - (2-aminoethyl) glycine chain to wich the nucleobase is tethered through an amide bond to the central amino group. Such a highly hydrophobic system is of course of low solubility and consequently of diminished cell 4

18 permeation ability. Compounds B and C represent a new polyamide backbone where the carboxylic moiety is replaced by phosphono groups to attain a more hydrophilic backbone resistant to nucleases degradation 19. In contrast to B, compound C consists of a delta hydroxy acid backbone. This allows chain elongation by methods adapted from oligonucleotide solid phase synthesis. Structures D-F are derived from a dipeptide (glycylglycine) segment, where the side chains bearing the nucleobases were placed at position C-1 via various spacers. The intervening alkyl bridge in compounds D, E, and F are methylene 20, ethylene 21 and methyl ketone respectively. Structure G is a true PA analogue bearing nucleobases linked through an acetamido moiety. Structure G is also a chiral PA bearing peptide side chains 22. It has been indicated in the literature that a stereogenic centers along the PA oligomer affect the handness of the PA-DA helix (L-lys and L-asp induced a left handed helix while D-lys and D-glu induced a preferred right handed helix). In general it can be said that the type of amino acid side chain and its chiral configuration affect the PA-DA duplex stability. In order to increase hybridization affinity, the notion is to form a more conformationally constrained molecule as in the case of inborn D and LA. The ability of PAs to strongly bind DA has been interpreted as a result of the constrained flexibility in its structure, since too flexible as well as too rigid analogs show very poor binding properties. Following these guidelines many PA analogs containing structures based on a cyclic scaffold such as prolyl, pyrrolidine, cyclopentyl, and picolyl (for general reviews see Ref. 23;24) have been described (fig 3) with the aim of achieving the desired balance between rigidity and flexibility. In this context, the stereochemistry is a very important feature since different stereoisomers have different abilities to adopt the conformation required for optimal DA or RA binding. Compound J is based on a glycine prolyl backbone 25 and consists of two chiral synthons. The cis stereomers exhibit a high binding propensity while the trans stereomers did not show any melting curve. Aminoprolyl PA (appa-compound K) 26 consists also of two chiral centers. o binding of homochiral oligomer was found, while mono-substitution stabilizes PA:DA duplexes. 5

19 Figure 3: PA structures based on cyclic scaffold Pyrrolidine PA (L) 27 consists of one asymmetric center. This compound destabilized binding to both complementary RA and DA. Aminoethylpyrrolidinone PA 28 (aepone-pa (M)) stabilized PA 2 -DA triplexes and destabilized poly-ra triplexes. In the case of cyclopentyl PA (cppa ()) 29, the data indicated that the Tm of most cppa-da duplexes (where the 1R,2S isomer is the predominant isomer) is higher 6

20 than the corresponding control PA, whereas Pipecolyl PAII (compound - pippa, 2S,4S) 31 significantly impairs PA-DA formation As mentioned previously, one of the disadvantages of the neutral PA units is their poor water solubility. In order to overcome this hurdle, many attempts were carried out in the search for a water soluble molecule. The rationale was to introduce a positive charge to the PA chain. This positive charge is thought, in addition, to potentially improve the cellular uptake. It is well documented in the literature that cationic residues confer several pharmacological advantages to many substances as: a. improved pharmacokinetics parameters in circulation, b. enhanced cell permeation and c. increased BBB transport properties. The introduction of a positive charge into a PA skeleton was implemented via five structural modes: a) Functional backbone: 3-4 positively charged amino acids such as lysine or arginine attached at the distal end of the PA chain, b) Chiral PA associated with cationic amino acids side chains as building blocks (see Fig 2 compound G), c) Ethylene linker bridging the nucleobase to the backbone (instead of an acetamide linker) as is depicted in fig 4 (compound 9 ethpa), d) Pyrrolidine and piperidine derived PA (fig 4), and e) Polyamine derived PA. Regarding (a), the introduction of 2-3 L (D) lysine or arginine suffers from several disadvantages: 1) As part of the antisense entity they might affect specificity and binding properties. 2) In vivo they may be subjected to proteolytic cleavage. 3) D-lys or arg are assumed to be toxic. 4) L-lys or arg are subjected in vivo to racemization. Regarding (b), it has been shown that a single insertion of an amino acid side chain to the PA backbone decreases, in general, binding of PA decamer (parallel and antiparallel mode) to DA or RA. In the case of arginine the observed Tm for parallel and antiparallel orientation of PA-DA duplex was -5 0 C and -2 0 C respectively, and -5 0 C for antiparallel PA-RA duplex. Similar results were observed with stereogenic lysine side chain 32. n the other hand Lys-based PA 33 and Arg-based PA 34 were shown to internalize cells efficiently. It was also 7

21 proposed that D aminoacid-containing PAs give rise to the formation of right handed structure when bound to its achiral complementary PA (antiparallel mode) while L-aminoacid containing PA give rise to a left handed duplex 35. Regarding (c) it was shown that insertion of a single unit destabilized PA/DA and PA/RA duplexes. Regarding (d), structures of cationic PA (including pyrrolidine and piperidine derived PA) are summarized in figure 4: Figure 4: Cationic derived PA The introduction of a pyrrolidyl unit (fig 4) stems from the notion that imparting structural rigidity to the PA backbone may improve its affinity. However, the 8

22 concomitant chirality may affect the selectivity. This is not the case for the aminoethyl prolyl PA (aep-pa) compound 2 (fig 4) where both, the 2S,4S and the 2S,4R isomers enhanced the stability of DA:PA 2 triplex by 5-8 degrees per modified unit. However, in the presence of a mismatch, the complex failed to show a sigmoidal transition which is probably partially due to the stringency of the structure 37. Examining compound 2 in PA:DA duplex revealed that thermal stability and parallel/antiparallel direction is controlled by nucleobases (purine/pyrimidine). In the case of antiparallel orientation, 2S,4S and 2R,4S diastereoisomers increased duplex stability, while in parallel orientation only 2S,4S configuration enforced duplex stability. In the case of the homopyrimidine PA sequence (T 8 ) comprising of a single T unit derived from compound 1 (fig 4 pyrrolidine PAII) it was shown that compared to the control sequence, the Tm's for the 2S,4S and 2R,4S configurations in the PA 2 :DA triplex are shifted by and respectively. Thus, it seems that the binding efficiency of compound 1 incorporated in the oligomer is dictated by the stereochemistry of its C2 center 43. This is in contrast to the behavior of compound 2 (fig 4). Incorporation of a single monomer of 3S,5R- pyrrolidine-2-one and 3R,5Spyrrolidine (fig 4, analog 3 (PA-I)) into a PA oligomer results in destabilization of the DA-PA and RA-PA duplexes. In the case of aminoethyl pipecolyl units (fig 4, aepip-pa 6) stabilization of a triplex formed by the homopolymer (T 8 ) PA 2 :DA depends on the position of a single inserted modified unit. A single modification in the middle or at the terminus did not affect the thermal stability of the 2S,5R isomer and decreased the Tm of the 2R,5S isomer (at terminus) by 10 0 C. n the other hand, a single modification at the C terminus of the poly T oligomer increased the thermal stability by Tm=+4 0 C and C for the 2S,5R and 2R,5S respectively. Increasing the number of aepippa to 2, enhanced the Tm by Tm = C; an effect that was position dependent. Interestingly, backbone extended pyrrolidine 2S,4S PA(bep) (fig 4, compound 8), exhibits a preferential binding selectivity to RA. The high binding affinity is exerted in the aeg-beppa alternating mode of PA 2 :RA triplex, exhibiting a Tm 9

23 value of C. Single insertion of bep-pa (T) at the or C terminus of the homopolymer aegt 8 results in binding to DA with only slight Tm changes (-2 0 C for the C terminus and +2 0 C for the terminus). The unusual stability of the hybrid between compound 5 (fig 4) and D can be partly attributed to the positive ly charged protonated hydrazine nitrogen atom on the PA, as the hybrid binding is ph sensitive exerting improved binding properties at lower ph. Finally, regarding (e) (see page 10, Polyamine derived PA), a multistep reaction route for the preparation of PamA incorporating a thymine base was previously described 38. The Tm value of a single secondary amine placed in an aegpa decamer did not destabilize PA:DA and PA:RA duplexes. ur approach is synthetically simpler and allows introducing all four natural bases. From a deep insight at the positively charged PA units presented in fig 4, it is apparent that all of them (except ethpa, comp. 9), impart a basic drawback related to the unit's chirality. We can infer that some of the different isomers bear opposite effects. Some binds preferentially to RA while the others to DA; some increase Tm values whereas others decrease Tm values. This inconsistency on their behavior encouraged us to return to the synthesis of the four ethpa monomers. We are aware of the fact, that this modification increases the flexibility of the side chain, compared to the methyleneamido link in the aegpa, and consequently affects the stability of the duplex and the ease of its formation. In order to overcome this hurdle, units that improve the hybridization may be introduced. To this aim two supporting elements were applied. ne is the 2,6 diaminopurine (DAP) and the second is the intercalatoria agents. 2,6-Diaminopurine (DAP) is a naturally occurring nucleobase that widely replaces adenine in D 39. Since a diaminopurine-thymine duplex resembles a guaninecytosine base pair in the sense of forming three hydrogen bonds (fig 5), it has been shown that incorporation of diaminopurine into short DA oligomers increases the thermal stability of the duplex by 0 2 o C per DAP-T base pair

24 Thus a similar increased affinity upon incorporation of diaminopurine could be expected for PA nucleic acid complexes. H 2 G H C H H 2 DAP H T H H H 2 H H 2 Figure 5: Hydrogen bonding between DAP/T ; G/C The second innovative approach is to enhance duplex binding (PA-DA) by the introduction of an intercalator module into the PA unit. A schematic presentation of the combined PA Intercalative agents is shown in Fig. 6: Figure 6: Intercalator between PA-DA Duplex The designed intercalators based on a polyamide chain are illustrated in Figure 7. In these constructs the nucleobase of a PA building block was replaced by an intercalator residue. In compound E1 the positive charge on the backbone unit is maintained (tert-amine) while this is not the case with compound E1A where the linkage between the lateral intercalator chain and the backbone is formed via an amide bond. 11

25 Intercalator Intercalator (CH 2 )n (CH 2 )n H 2 H 2 C C H 2 C H 2 CH H 2 H 2 C C H 2 C H 2 CH E1 E1A Figure 7: Interchalator connection modes on the PA backbone In the cases described in the literature the intercalators are conjugate to internal amine of the polyamide backbone via a short linker as is shown in Fig. 7. This may on the one hand attenuate binding due to the replacement of the complementary base by a residue void of hydrogen bonding features; however on the other hand, replacement of the complementary base for the intercalator moiety with improved stacking properties may confer a better binding property. The resultant of these two modes of action contributes to the intercalator s stabilization or destabilization feature (See model A). ur approach circumvents this hurdle since the intercalator in our model is embedded between the bases and thus, does not substitute for a nucleobase and subsequently does not interfere with hydrogen bonding (Model B). Due to that it is expected that intercalators based on PA structure designed as is depicted in model B will significantly contribute to duplex and triplex stability. Model A Model B Intercalator Intercalator Figure 8: Intercalator Model A & Model B 12

26 Since bis intercalators were found to stabilize the PA/DA duplex, the binding model portrayed in Fig. 8 seems to be feasible. The intercalators linked to the PA via the polyamide backbone units (See Fig. 7) at the distal end of the sequence are free to intercalate either as bis or tris structure. In order to avoid orientation problems a spacer can also be introduced. Biological Context varian cancer is the most common cause of death from gynecologic malignancy among women in the Western world. The high mortality associated with ovarian carcinoma reflects a difficulty in diagnosing the disease before the tumor has metastasized, due to asymptomatic progression at early stage. Recent studies revealed a cancer-preventive role for non-steroidal anti-inflammatory drugs (SAIDs), mediated through inhibition of cyclooxygenase (CX), the enzyme that converts AA to prostaglandins and thromboxanes. In our recent study on ovarian tumor cells, we found that PLA2 isoforms, were expressed in the majority of the specimens. f interest was the association of MMP-2 with activated-cpla2 and spla2-iia proteins. The spla2-iia isoform is sorted into caveolin-rich compartments where it is associated with HSPG glypican and internalized into the cell. Inside the cell, spla2-iia is localized to vesicular and perinuclear membranous sites where arachidonic acid is subsequently released. ur hypothesis is that silencing the glypican gene, responsible for the translocation of spla2-iia into the cell will prevent/reduce the dissemination of this tumor. 13

27 Hypothesis and Research objective The hypothesis of this research is summarized by the following aspects: Positively charged agents tethered to PA sequences will increase its solubility and cell uptake levels. Binding anchors such as; interchalators (anthraquinone, anthracene and pyrene) and dap will enhance the total hybridization of PA:DA duplexes. The objective of the present research is to prepare new analogues of PA which inclined to increase levels of solubility, cell permeation and hybridization with the complementary glypican-1 mra. In order to attain these goals we tend to implant on the PA backbone: a. Cationic units (to increase the solubility and cell permeation). b. Intercalator units (to enhance the hybridization). c. Diaminopurine (to form three hydrogen bonds with T instead of two from A). In this context, our specific aims were: 1. To prepare PA backbone units {H 2 CH 2 CH 2 H(R)CH 2 CH} bearing residue of: Positively charged elements Intercalators such as anthraquinone, pyrene and anthracene DAP 2. To prepare PAs bearing the modifying units against the mra of glypican Synthesis of positively charged PA directly on solid support. 14

28 EXPERIMETAL 15

29 MR: MR data were collected on Varian VXR-300 MHz spectrometer equipped with a 5- mm switchable probe. Data were processed using the VMR software. MR abbreviations are as follows: s (singlet), d (doublet), t (triplet), bt (broad triplet), q (quartet), m (multiplet), br (broad). Electrospray Ionization Mass Spectrometry (ESI-MS): Electrospray ionization mass spectrometry was measured on a ThermoQuest Finnigan LCQ- Duo in the positive ion mode. Data were processed using ThermoQuest Finnigan s Xcalibur Biomass Calculation and Deconvolution software. UV/Visible: Uv/Visible spectrophotometric data were collected on an Ultrospec 2100 Pro Spectrophotometer. Data were processed using Swift II Wavescan TM software. 16

30 BC & t-bu Protected Monomers - Method A 1-(tert-Butyloxycarbonyl)ethylenediamine (2) A solution of di-tert-butyl dicarbonate (6.1 g, 28 mmol) in dichloromethane (400 ml) was added dropwise to a solution of ethylenediamine (11.2 ml, mmol) in dichloromethane (50 ml) over 6 h with vigorous stirring. Stirring was continued for 24 h at room temperature. After concentration to an oily residue, the reaction mixture was dissolved in aqueous sodium carbonate (2 M, 300 ml) and extracted with dichloromethane (2 300 ml). The organic layer was dried (anhydrous MgS 4 ) and the solvent evaporated under reduced pressure to yield the desired product as a colorless viscous liquid. Yield: 95% ESI MS: m/z: calcd: H MR (CDCl 3 ): 4.89 (br, 1H, H), 3.18 (dt, 2H, CH 2 CH 2 H), 2.80 (t, 2H, CH 2 CH 2 H), 1.60 (br, 2H, H), 1.44 (s, 9H, tbu). -[(tert-butoxycarbonyl)-aminoethylamine]-glycine-tert-butyl ester (3) 1-(tert-butoxycarbonyl)-aminoethylamine (16 g, 99.8 mmol) was dissolved in 200 ml methylene chloride together with 2 equivalents (34.8 ml, mmol) of diisopropyethylamine (DIEA). After cooling the mixture to 0 C, 1 equivalent (14.75 ml, 99.8 mmol) of TBBA (tbu-bromoacetate) was added dropwise over a period of 6 hours. The reaction mixture was stirred overnight.the precipitate was filtrated off and the solvents were removed under vacuum. The resulting gum was distributed between 300 ml H 2 (DDW) and 300 ml dichloromethane. The aqueous phase was washed with dichloromethane (4 * 200 ml). The organic fractions were pooled and dried over sodium sulfate. After filtering off the solids the dichloromethane was evaporated to dryness. The desired product was achieved after silica gel purification (ethyl acetate- 10%MetH in ethyl acetate). Yield: 73% ESI MS: m/z: calcd:

31 1H MR (CDCl 3 ): 5.05 (br s, 1H, H), 3.28 (s, 2H, CH 2 C), 3.18 (dt, 2H, CH 2 CH 2 H), 2.80 (t, 2H, CH 2 CH 2 H), 1.46 (s, 18H, Boc + tbu). [(2-tert-Butoxycarbonylamino-ethyl)-(4-dimethylamino-butyryl)-amino]-acetic acid tert-butyl ester (6) A dimethylamino butyric acid hydrochloride (0.88gr, 5.25mmol) and 1ml diisopropylethyl amine was dissolved in 35ml DMF, the mixture was cooled to 0 0 C under icebath then DCC ( 1.62gr, 7.87mmol ) and HBT ( 0.78gr, 5.89mmol) were added. the mixture was stirred under cooling for 30 minutes, then -{(tertbutoxycarbonyl)-aminoethylamine}-glycine-tert-butyl ester (1.2gr, 4.38mmol) in 7ml DMF was added and the mixture was lefted to overnight and monitored by TLC which show slower mobility for the product than back bone. Then the white precipitate was removed by filtration, while the filtrate was evaporated under reduced vacuum. The resulted residue was dissolved in DCM 60 ml and washed with 10% ahc 3 2x50ml, 2x50ml water. The organic layer was dried over sodium sulphate and evaporated to give 1.36 gr crude product which was purified by 10%MeH in EtAc to give 0.72gr. Yield: 42% ESI-MS: m/z: 388 calc. 387 'H MR (DMS-d 6 ): 5.05 (br s, 1H, H), 3.28 (s, 2H, CH 2 C), 3.18 (dt, 2H, CH 2 CH 2 H), 2.80 (t, 2H, CH 2 CH 2 H), 1.46 (s, 18H, Boc + tbu), 2.36 (t, 2H, CH 2 CH 2 CH 2 C),1.67 (P, 2H, CH 2 CH 2 CH 2 C), 2.18 (t, 2H, CH 2 CH 2 CH 2 C), 2.27 (s, 6H, CH 3 ). 1-{(2-Carboxyethyl)amino}anthraquinone ( 4 ) 1-Aminoanthraquinone (2.5gr, 10 mmol), AlCl 3 (1.25gr, 10mmol) and ethyl acrylate was (5ml, 46mmol) were refluxed in 50 ml dry dioxane for 48 hr.following the reflux, 125ml 1 HCl in order to decompose the excess of allominium chloride. The organic substance was extracted with several washes by DCM and dried over magnesium solphate. After evaporation the solvent under reduced vaccum 50 ml KH in methanol was added and the reaction mixture was left in r.t for a week. 200 ml 1 HCl were added and DCM was used for extraction. The combined organic 18

32 layer was dried over magnesium solphate and the solvent evaporated to dryness to give red product. Yield: 83% ESI MS:m/z: 296 calc. 295 'H MR (DMS-d 6 ): (m, 4H, AQ), (m, 3H, AQ), 4.2 (t, 1H, H- AQ), 3.34 (q, 2H, HCH 2 CH 2 ), 2.46 (q, 2H, HCH 2 CH 2 ). {(2-te rt-butoxycarbonylamino-ethyl)-[3-(9,10-dioxo-9,10-dihydro-anthracen-1- ylamino)-propionyl]-amino}-acetic acid tert-butyl ester (5) 1-{(2-Carboxyethyl) amino}anthraquinone (0.13gr, 0.45mmol) was suspended in DMF 5ml under cooling with ice-bath to 0 0 C. DCC (0.14gr, 0.675mmol) and HBT (0.067gr, 0.5mmol) were added and the mixture was stirred at 0 0 C for 30 min, then - {(tert-butoxycarbonyl)-aminoethylamine}-glycine-tert-butyl ester (0.122gr, 0.45mmol) was added. The reaction mixture stirred for 48hr at r.t, then the white precipitate was removed by filtration and the filtrate was evaporated under reduced vacuum. The resulted gum was dissolved in 20 ml DCM and washed with 10% ahc 3 2x20m, 2x20ml water. The organic layer was dried over sodium solphate and evaporated to give 0.28gr red crude product which purified by HPLC: eluent; 5-80% methanol in water. Yield: 72% ESI-MS: m/z: 552 calc. 551 'H MR (DMS-d 6 ): (m, 4H, AQ), (m, 3H, AQ), 4.55 (t, 1H, H-AQ), 3.34 (q, 2H, HCH 2 CH 2 ), 2.46 (q, 2H, HCH 2 CH 2 ), 5.05 (br s, 1H, H), 3.28 (s, 2H, CH 2 C), 3.18 (dt, 2H, CH 2 CH 2 H), 2.80 (t, 2H, CH 2 CH 2 H), 1.46 (s, 18H, Boc + tbu). [(2-tert-Butoxycarbonylamino-ethyl)-methyl-amino]-acetic acid tert-butyl ester (7) To a -{(tert-butoxycarbonyl)-aminoethylamine}-glycine-tertbutyl ester (0.25gr, 0.89mmol) formaldehyde 37% in water ( 0.2ml, 7.12mmol) was added and heated to reflux for overnight. The reaction was monitored by TLC, and then the mixture was 19

33 evaporated to remove water and dissolved in ethanol 10ml, sodium cyanoborohydride (0.17gr, 2.7mmol) was added by cooling for 30 min then warmed to room temp and lefted to overnight and monitored by TLC which shows faster mobility to the product vs. starting material. After reaction complete 0.1M HCl was added drop-wise until acidic PH, white precipitate was removed by filtration.the filtrate was evaporated under reduced vacuum to give 0.35gr crude product, which purified by column chromotography; eluent: EtAc. Yield: 94% ESI-MS: m/z: 289 calc H MR (CDCl 3 ): 5.05 (br s, 1H, H), 3.28 (s, 2H, CH 2 C), 3.18 (dt, 2H, CH 2 CH 2 H), 2.80 (t, 2H, CH 2 CH 2 H), 2.27 (s, 3H, CH 3 ), 1.46 (s, 18H, Boc + tbu). Benzyl & Phtalimide Protected Monomers - Method B [2-(phthalimide)-ethylamino]-acetic acid benzyl ester (8) Glycine benzyl ester p-toluene sulphonate (5gr, 15mmol) was suspended in 30ml DCM, triethylamine (8 ml, 30mmol) was added. The mixture was stirred vigorously under reflux. -(2-Bromoethyl) phthalimide (3.77gr, 15mmol) dissolved in 15 ml DCM was added drop-wise over 6 hr. The mixture left for overnight, then washed 3x100 ml water, the organic layer dried over sodium sulphate and solvent removed under reduced vacuum to give yellowish oily residue which purified with DCM/EtAc (1:1). Yield: 82% ESI MS: m/z: 339 calc. 338 'H MR (DMS-d 6 ): 5.34 (s, 2H, CH 2- Ph), 7.19 (m, 5H, Ph), 3.51 (d, 2H, CH 2 H), 2.88 (t, 2H, HCH 2 CH 2 ), 3.75 (t, 2H, HCH 2 CH 2 ), (s, 4H, phtalimide). 20

34 {(4-Dimethylamino-butyryl)-[2-(phthalimide)-ethyl]-amino}-acetic acid benzyl ester (9) Dimethylaminobutyric acid hydrochloride (0.13gr, 13.53mmol) was dissolved in DMF, ml DIEA were added. The mixture was cooled under ice bath. To the solution HBT (2.0gr, mmol) and DCC (3.0gr, 14.76mmol) were added. The reaction mixture was stirred for 30 min, then [2-(phthalimide)-ethylamino]-acetic acid benzyl ester (4.15gr, 12.3mmol) dissolved in ml DMF was added in portions. The reaction temperature was elevated to r.t. and left for 48 hr. Then the solvent was removed under reduced vacuum, the residue was dissolved in DCM 100 ml and washed with saturated sodium bicarbonate 3x 100 ml. The organic layer was dried over sodium sulphate and the solvent removed by evaporation to give yellow oil. Yield: 74% ESI MS: m/z: 452 calc. 451 'H MR (DMS-d 6 ): 2.36 (t, 2H, CH 2 CH 2 CH 2 C),1.67 (P, 2H, CH 2 CH 2 CH 2 C), 2.18 (t, 2H, CH 2 CH 2 CH 2 C), 2.27 (s, 6H, CH 3 ), 5.34 (s, 2H, CH 2- Ph), 7.19 (m, 5H, Ph), 3.51 (d, 2H, CH 2 ), 2.88 (t, 2H, HCH 2 CH 2 ), 3.75 (t, 2H, HCH 2 CH 2 ), (s, 4H, phtalimide). {[2-(phthalimide)-ethyl]-[2-(tert-buthoxycarbonylamino)-acetyl]-amino}-acetic acid benzyl ester (12) BC-Gly-H (0.32gr, 1.77mmol) was dissolved in DMF10 ml and cooled under ice bath to 0C 0. To the mixture DCC (0.45gr,2.66mmol) and HBT (0.36gr, 2.66mmol) were added and stirred for 30 min at 0C 0, then [2-(phthalimide)-ethylamino]-acetic acid benzyl ester (0.5gr, 1.47mmol) was added in portions.the reaction mixture was elevated to r.t. and left for 48hr and monitored by TLC. Then DMF removed by reduced pressure. The residue was dissolved in DCM ml and washed with 3xml DDW, the organic layer was dried over sodium sulphate and evaporated to remove the solvent to give white solid. Yield: 64% ESI MS: m/z: 496 calc

35 'H MR (DMS-d 6 ): 5.34 (s, 2H, CH 2 Ph), 7.19 (m, 5H, Ph), 3.51 (d, 2H, CH 2 ), 2.88 (t, 2H, HCH 2 CH 2 ), 3.75 (t, 2H, HCH 2 CH 2 ), (s, 4H, phtalimide), 3.85 (s, 2H, CCH 2 H), 1.46 (s, 18H, tbu). {(2-Amino-acetyl)-[2-(phthalimide)-ethyl]-amino}-acetic acid benzyl ester (13) {[2-(phthalimide)-ethyl]-[2-(tert-buthoxycarbonylamino)-acetyl]-amino}-acetic acid benzyl ester (0.4gr, 0.8mmol) was dissolved in 50% TFA/DCM solution ml at r.t for 5hr and to cleave Fmoc group. The reaction monitored by TLC. The product show slower mobility Vs. the starting compound. The DCM removed under reduced pressure to give 0.92mmol. Yield: 88% ESI MS: m/z: 396 calc. 395,'-bis(boc)-S-methylisothiourea (11) S-methylisothiourea sulphate (2.78gr,10mmol) was dissolved in a mixture of water 30ml and dioxane 30ml followed by addition of 1M aq, ah solution (20ml, 20mmol) and di-tert-butyl dicarbonate (11.62gr, 50 mmol).the reaction mixture was stirred vigorously at r.t. for overnight. DCM 30 ml was added and the aqueous phase was extracted twice more with DCM. The combined organic layers was washed with diluted HCl (0.02M, 3x) to remove the monobc-s-methylisothiourea.the organic layer was then washed with saturated H 4 Cl 2x50 ml and dried over sodium solphate and evaporated to give a white solid.the solid was suspended twice in water 200 ml at approximately 50 C 0, shaken and filtered. It was then dried under reduced vacuum over P 2 5 to give the title compound as a white solid. Yield: 95% ESI MS: m/z: 191 calc. 190 'H MR (CDCl 3 ): δ 1.49 (s, 9H), 1.51 (s, 9H), 2.38 (s, 3H), (s, 1H) {(2-(1,2-Bis(tert-butoxycarbonyl)guanidino)-glycyl)-[2-(phthalimide)-ethyl]- amino}-acetic acid benzyl ester (14) 22

36 {(2-Amino-acetyl)-[2-(phthalimide)-ethyl]-amino}-acetic acid benzyl ester (0.36gr, 0.92mmol) was dissolved in DCM ml, triethylamine (170ml,1.84mmol) and,'- bis(boc)-s-methylisothiourea (0.17gr,0.92mmol) were added and stirred at r.t. for overnight and monitored by TLC. Yield: 56% ESI MS: m/z: 452 calc. 451 'H MR (DMS-d 6 ): 5.34 (s, 2H, CH 2 Ph), 7.19 (m, 5H, Ph), 3.51 (d, 2H, CH 2 ), 2.88 (t, 2H, HCH 2 CH 2 ), 3.75 (t, 2H, HCH 2 CH 2 ), (s, 4H, phtalimide), 3.88 (s, 2H, CCH 2 H), 1.46 (s, 18H, BC). [[2-(phthalimide)-ethyl]-[pyrene-4-yl-butyryl)-amino]-acetic acid benzyl ester (10) 1-pyrene butyric acid (0.51gr, 1.77mmol) was dissolved in DMF ml, DCC (0.54gr, 2.66mmol) and HBT (0.36gr, 2.66mmol) were added. the mixture cooled under ice bath and stirred at 0C 0 for 30 min. [2-(phthalimide)-ethylamino]-acetic acid benzyl ester (0.5gr, 1.48mmol) in DMF ml was added in portions,the reaction mixture was elevated to r.t and stirred for 48 hr, then DMF removed under reduced pressure,the residue was dissolved in DCM ml and washed with 3xml DDW. The organic layer was dried over sodium solphate and evaporated to remove the solvent to the desired product which purified by silica gel chromatography with. Yield: 68% ESI MS: m/z: 452 calc. 451 'H MR (DMS-d 6 ): 5.34 (s, 2H, CH 2- Ph), 7.19 (m, 5H, Ph), 3.51 (d, 2H, CH 2 H), 2.88 (t, 2H, HCH 2 CH 2 ), 3.75 (t, 2H, HCH 2 CH 2 ), (s, 4H, phtalimide), 2.18 (t, 2H, CCH 2 CH 2 CH 2 ), 1.92 (q, 2H, CCH 2 CH 2 CH 2 ), 2.99 (t, 2H, CCH 2 CH 2 CH 2 ), (m, 9H, Pyr). Tert-butyl ( 15 ) Bis-2,6-[Bis(tert-butoxycarbonyl)amino]-9H-purine-9-carboxylate To a 100 ml Ar flushed flask equipped with a magnetic stir bar containing 2,6- diaminpurine (1gr, 6.66mmol) and DMAP (0.08gr,0.66mmol) was added dry THF 50 23

37 ml. To the stirred suspension was added BC 2 (7.3gr, 33.3mmol) under an Ar atmosphere. The reaction mixture was stirred overnight at room temperature, at which point TLC analysis indicated the presence of single product. The excess amount of THF was removed to give gr as yellow oil, which purified on silica gel with hexane/ethylacetate (7:3). Yield: ESI MS: m/z: 651 calc. 650 'H MR (DMS-d 6 ): 7.67 (s, 1H, H-C 8 ), 1.46 (s, 18H, 2 ), 1.50 (s, 18H, 6 ), 1.48 (s, 9H, 9 ). Tetra-BC- 2,6-diaminopurine (16) To a solution of 18 (4.29gr, 6.54mmol) dissolved in methanol 80 ml was added saturated aq. ahc 3 40ml. The turbid solution was stirred at 50C 0 for 1hr, at which point clean conversion to quat-bc protected diaminopurine was observed by TLC. After evaporation of methanol, water 50 ml was added to the suspension and the aqueous layer was extracted with DCM 3x100 ml. The organic layer was dried with a 2 S 4, filtered and evaporated to give gr as white solid which purified on silica gel with ethyl acetate. Yield: 96% ESI MS: m/z: 551 calc. 550 'H MR (DMS-d 6 ): 7.67 (s, 1H, H-C 8 ), 1.46 (s, 18H, 2 ), 1.50 (s, 18H, 6 ). Benzyl 2-Bis-{2,6-Bis(tert-butoxycarbonyl)amino]-9H-9-purine-yl}-acetate (17) To a solution of 19 ( 3.6gr, 6.28mmol) in dry THF 60 ml cooled under ice bath for 15 min. was added ah ( 0.23gr, 9.42mmol) under an argon atmosphere whilst stirring.to a rapidly stirred mixture was then added dropwise over 10 min. benzyl bromoacetate ( 1.2ml,7.53mmol) and DMAP (0.03gr,0.15mmol). After complete addition, the reaction was stirred overnight whilst warming to room temperature. The reaction mixture was then quenched by the addition of water (20ml, 19mmol). The solvent THF was removed and the residue was then redissolved in DCM 250ml and washed with water 3x80ml. After evaporation of THF light yellow oil was obtained, 24

38 which was purified by silica gel chromatography with hexane/ethyl acetate (6:4) to give gr as pale-yellow oil. Yield: 72% ESI MS: m/z: 699 calc. 698 'H MR (DMS-d 6 ): 7.67 (s, 1H, H-C 8 ), 1.46 (s, 18H, 2 ), 1.50 (s, 18H, 6 ), 4.8 (s, 2H, 9 -CH 2 ), 5.34 (s, 2H, CH 2- Ph), 7.19 (m, 5H, Ph). 2-{2,6 Bis-[Bis(tert-btoxycarbonyl)amino]-9H-purine-9-yl}-acetic acid (18) To a suspension of 21 (3.11gr, 4.55mmol) in methanol 100 ml was added 5% Pd/C (0.25gr) under an argon atmosphere, and the mixture was hydrogenated until the substrate disappeared, monitored by TLC about 8-12 hr. The reaction mixture filtered through a pad of celite, the solvent was evaporated to give gr as white solid, which purified on silica gel with hexane/ethyl acetate (6:4). Yield: 98% ESI MS: m/z: 609 calc. 608 'H MR (DMS-d 6 ): 7.67 (s, 1H, H-C 8 ), 1.46 (s, 18H, 2 ), 1.50 (s, 18H, 6 ), 4.8 (s, 2H, 9 -CH 2 ), 5.34 (s, 2H, CH 2 C). {2-{2,6-Bis-[Bis(te rt-btoxycarbonyl)amino]-9h-purine-9-yl}-acetyl-[2- (phthalimide)-ethyl]-amino}-acetic acid benzyl ester (19) 2-{2,6 Bis-[Bis(tert-btoxycarbonyl)amino]-9H-purine-9-yl}-acetic acid ( 0.89gr, 1.48mmol) was dissolved in DMF ml and cooled to 0C 0 under ice bath. HBT (0.36gr, 2.66mmol) and DCC (0.54gr, 2.66mmol) were added and the mixture was stirred at 0C 0 for 30 min, then [2-(phthalimide)-ethylamino]-acetic acid benzyl ester (0.5gr, 1.48mmol) dissolved in DMF ml was added in portions and stirred for additional 30 min. at 0C 0 then the reaction mixture elevated to r.t. and stirred for 48 hr monitored by TLC. After evaporation of DMF the residue was redissolved in DCM ml and washed with DDW 3xml.The organic layer was dried over sodium solfate and evaporated to remove the solvent to give the desired product as a white solid gr. Yield: 53% 25

39 ESI MS: m/z: 929 calc.928 'H MR (DMS-d 6 ): 5.34 (s, 2H, CH 2- Ph), 7.19 (m, 5H, Ph), 3.51 (d, 2H, CH 2 H), 2.88 (t, 2H, HCH 2 CH 2 ), 3.75 (t, 2H, HCH 2 CH 2 ), (s, 4H, phtalimide), 7.67 (s, 1H, H-C 8 ), 1.46 (s, 18H, 2 ), 1.50 (s, 18H, 6 ), 4.8 (s, 2H, 9 -CH 2 ), 5.34 (s, 2H, CH 2- Ph), 7.19 (m, 5H, Ph). Cleavage of the benzyl protecting group under hydrogenation (9, 10, 14, 19) to give (20, 21, 22, 23) respectively General procedure The back bone carboxylic acids derivatives were dissolved in 100 ml methanol, about of 0.30gr Pd/c 5% was added and hydrogenated under 30 psi for overnight, and then the mixture was filtrated through a pad of celite. The filtrate was evaporated under reduced pressure to give white solid, which purified on silica gel with hexane/ethyl acetate (6:4). Yield: 94-98% Preparation of Lysine derivative Monomers α -FMC- ε -Dimethyl Lysine (24) To a stirred solution of α -FMC-Lysine hydrochloride (1gr, 2.5mmol) on 50 ml Ethanol under ice bath, 37%aqueous formaldehyde (0.43ml, 5.5mmol) was added. After stirring for 15 min, sodium cyanoborohydride (0.48gr, 7.5mmol) was added while the temperature of a reaction mixture kept at 0C 0 after 15 min formaldehyde and sodium cyanoborohydride treatment were repeated. The reaction mixture was stirred for additional 2hr. after TLC (MeH/H 2, 95:5, Rf ~0.4) detection showed that reaction was complete, 1M HCl was added drop-wise till the solution was acidic, and some white precipitate formed. The mixture was filtrated to remove the precipitate. Crude product was obtained after the solvent of filtrate was removed by evaporation under reduced pressure and then purified with silica gel column using gradient (0-5% H 2 / MeH) as eluent, to give 980mg pure product. 26

40 Yield: 94% ESI MS: m/z: 397 calc. 396 'H MR (DMS-d 6 ): (m, 8H, Ar-H/Fmoc), 7.1 (d, 1H, H), 4.2 (m, 3H, CH/Lys, CH/Fmoc), (m, 1H), (t, 2H, CH 2 /Lys), (m, 2H, CH 2 /Lys), (m, 2H, CH 2 /Lys), (m, 2H, CH 2 /Lys), 2.2 (s, 6H,Me 2 ). α -FMC- ε -carboxy butyric Pyrene Lysine (26) 1-pyrene carboxylic acid (1.0gr, 3.47mmol) was dissolved in 8ml DMF, HBT (0.7gr, 5.2mmol) and DCC (1.1gr, 5.2mmol) were added. The mixture was stirred at r.t for overnight. The white precipitate was removed by filtration, the mixture was cooled to 0C 0 under ice bath for 30 min. then α -FMC-Lysine hydrochloride (1.4gr,5.2mmol) dissolved in DMF 2ml with DIEA(0.9ml,5.2mmol) were added in portions. The reaction mixture was elevated to r.t and stirred for 48hr.The mixture filtered again and evaporated to remove DMF. The residue received after purification by silica gel with EtAc as eluent. Yield: 78% ESI MS: m/z: calc. 368 'H MR (DMS-d 6 ): (m, 8H, Ar-H/Fmoc), 7.1 (d, 1H, H), 4.2 (m, 3H, CH/Lys, CH/Fmoc), (m, 1H), (t, 2H, CH 2 /Lys), (m, 2H, CH 2 /Lys), (m, 2H, CH 2 /Lys), (m, 2H, CH 2 /Lys), 2.18 (t, 2H, CCH 2 CH 2 CH 2 ), 1.92 (q, 2H, CCH 2 CH 2 CH 2 ), 2.99 (t, 2H, CCH 2 CH 2 CH 2 ), (m, 9H, Pyr). α -FMC- ε -carboxy-9-anthracene Lysine (26) Anthracene-9-carboxylic acid (1.0gr, 4.49mmol) was dissolved in 10ml DMF, HBT (0.92gr, 6.75mmol) and DCC (1.4gr, 6.75mmol) were added. The mixture was stirred at r.t for overnight. The white precipitate was removed by filtration, the mixture was cooled to 0C 0 under ice bath for 30 min, and then α -FMC-Lysine hydrochloride (2.73gr, 4.5mmol) dissolved in DMF 2ml with DIEA (1.2ml, 6.75mmol) were added in portions. The reaction mixture was elevated to r.t and stirred for 48hr.The mixture 27