Self-eliminating cleavable linkers

Abstract

The invention is directed to self-eliminating cleavable conjugates according to general formula (I) or (II) wherein AR is capable of an elimination reaction upon cleavage of CL.

Description

SELF-ELIMINATING CLEAVABLE LINKERSBACKGROUND

[0001] The present invention is directed to self-eliminating cleavable linkers, and their applications for bio-molecular detection such as proteins, DNA and RNA molecules. The cleavable linkers allow removing the reporter group such as fluorescent dyes after detection. The linkers disclosed herein comprises structural motif(s) that undergoes cascade of self-elimination reactions upon cleavage of the cleavable functional group. Since the linker components are eliminated, the end product of the cleavage reaction leaves no or very little scars on the proteins and DNA probes. This type of linkers also have potential applications in drug deliveries as well, such as antibody-drugs conjugates etc. for site specific deliveries.

[0002] Chemical linkers are used for plethora of applications such as in the detection of biomolecules (proteins, DNA, RNA, carbohydrate epitopes etc), solid phase synthesis of peptides and the nucleic acid molecules, as wells as in targeted drug delivery applications. On the other hand, cleavable linker are those that could be cleaved off at will by chemical, enzymatic, or photochemical means. In molecular detection, cleavable linkers allow removing reporter group. One of the most common cleavable linkers is disulfide. It can be cleaved off efficiently by treating with biocompatible reducing reagents such as TCEP, THPP etc.

[0003] Although this linker is widely used now, it suffers significant limitations due to its limited stability and the non-specific interaction with free thiols exist in biological samples. The disulfides and other chemically cleavable linkers also leave scars upon cleavage, which can be undesirable and detrimental to the subsequent steps.

[0004] US2024 / 132955 and US2023 / 407389 Al disclose a chemically different class of compounds as self-eliminating cleavable conjugates. Having a different structure, they have a different cleavage mechanism as those described this the present invention.

[0005] The compounds disclosed in US2024 / 132955 and US2023 / 407389 Al have limited storage stability (as they are disulfides), can undergo side reaction with biological materials due to interaction with thiol groups, and possess a slow self-elimination kinetics. Such linkers are useful in drug delivery application where slow kinetics is often desired property.

[0006] However, molecular detection applications, high stability and rapid kinetics of the cleavage reaction are important aspects. Accordingly there was a need for self-eliminating cleavable conjugates exhibiting high stability with instantaneous self-elimination kinetics.SUMMARY

[0007] Object of the invention are self-eliminating cleavable conjugates according to general formula I or IIWithCG: cleavable group comprising at least one residue selected from the group consisting ofwith Re-Rii: independently H, D, F, Cl, Br, substituted or unsubstituted alkyl groups having 1 to 10 caron atoms and Me = CH3.AR: aromatic 6 membered ringRM: recognizing moiety selected from the group consisting of DNA, antibody, nucleotideRi to Rs: independently H, alkyl with 1 to 10 carbon atoms, Cl, F, -OR, -SR, -NR2 or - NHR with R = alkyl having 1 to 10 carbon atomsSP: spacer unit selected from the group consisting of PEG, peptide chain or alkyl chain having 2 to 10 carbon atomsZ: -O- or -S-Y : linking functional group comprising at least one selected from the groups consisting of carbonate : -O(C=O)O-, carbamate: -NH(C=O)O-, thio-carbonate: -S(C=O)O-, phosphate: - OP(=O)(OH)O- or sulfonate: -OS(=O)2-O-.Label: fluorescent unit, for example as FAM, R6G, ROX, Cy5, Alexa dyes, or ATTO dyes

[0008] Preferable, AR is capable of 1,6 elimination reaction upon cleavage of CL.

[0009] For examples, in drug delivery applications, the disulfide linker could reduce effectiveness of the drugs due to chemical changes in drug molecules attributed to the thiol scars. In detecting of biomolecules, the thiol scars could be detrimental to the subsequent steps and processes due to increased thiol group accumulation in the system. The increased scars and thiol group accumulation can interact non-specifically with the incoming reagents in the subsequent steps.

[0010] Here we disclosed a series of cleavable linkers that circumvent above stated limitations. The linkers could be made of varying stability by choosing appropriate substituent groups adjacent to the cleavable functional group to prevent from side reaction in biological samples, but yet can be easily cleaved off by biocompatible reducing reagents. Soon the cleave step takes place the linker undergoes rapid one or more steps of self-elimination / self- immolation reaction or react with the media (such as with water to undergo hydrolytic dissociation) to eliminate the linker from the point of attachment.

[0011] The end product of the cleave reaction is native form of the probes or drug molecules - having no modification. The most importantly it does not leave reactive thiol (-SH) group as scars. Since the modem antibody and nucleic acids detection involves repetitive cycling steps, the linkers disclosed herein devoid any buildup of non-specific signals associated with the thiol build up. Apart from these, with appropriate substitution on the adjacent carbon atoms, such linker can be made more stable. And if desirable, a lesser stable linkers can also be made which can be useful in drug delivery applications where slow degradation of the linkers can a desirable attribute.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 shows probes with regular disulfide leaves thiol scars on the target after cleavage (c), which can interact non-specifically with incoming reagent in subsequent step (d). The probe molecules can also react with other thiol groups (e) exist in biological sample such as with the free thiol or disulfide group in protein molecules.

[0013] Figure 2 shows protein probes (e.g. labeled antibody) with disulfide linker can interact with the free thiol groups of the targets as well as non-target proteins (c). Also after cleave off the disulfide linker, the thiol scars produced on the protein molecules can interact with the incoming reagents in the subsequent steps (e).

[0014] Figure 3 shows selective examples of self-eliminating linkers. They undergo selfelimination upon treatment of TCEP or THPP, leaving little or no trace associated with the respective linkers.

[0015] Figure 4 shows generic structures of the self-eliminating disulfide linker with a chain consisting of (probe - carbamate - benzyl - oxymethyl - disulfide - spacer - fluorescence dye label).

[0016] Figure 5 shows generic structures of the self-eliminating linker, cleavable by phosphine based compounds: (probe - carbamate - benzyl - azidomethyl - spacer - fluorescence dye label) type self-eliminating cleavable linker, linker. The reduction of the azide group by phosphines triggers cleavage and instantaneous self-elimination.

[0017]

[0018] Figure 6 shows generic structure of self - eliminating cleavable linker consisting ethylene and double disulfide bridge (A) and a specific example (B).

[0019] Figure 7 shows generic structure of self - eliminating cleavable linker consisting ethylene and double disulfide bridge (A) and a specific example (B).

[0020] Figure 8 shows cleavable linker consisting of benzyl-carbonate-ethylene-BMS- spacer. Other aspects includes substituted ethylene disulfide or benzyl disulfide with attached spacer such as PEG spacer. The probes and the linker can be attached via carbonate or carbamate ester groups.

[0021] Figure 10 shows labeled reversible nucleotide terminators with self-eliminating linkers

[0022] Figure 11 shows linkers with activated forms, and structures with “clickable” functional groupsDETAILED DESCRIPTION

[0023] The figure 1 shows how the DNA probes with commercially available disulfide linker can leave reactive thiol scars (-SH) group upon cleavage and the same group could interact non- specifically with the incoming probes in subsequent steps. The same disulfide group can also interact non-specifically with other thiol containing biomolecules such as with proteins, causing undesirable chemical modification to the latter.

[0024] Similarly, with the protein or polypeptides (e.g. labeled antibody probes) the cleavable linkers with regular disulfide (Figure 2) can also undergo non-specific covalent interaction with other thiol containing proteins. Such interaction can cause significant background build up in image.

[0025] The above stated detrimental effects, but not limited to, can be eliminated by using stable linkers with self-elimination properties upon cleavage.

[0026] Here we disclose a number of cleavable linkers for probes labeling that undergo cascade of reactions upon cleavage leaving no “thiol scars” on the probes. They contain a cleavable functional group (e.g. disulfide, - azidomethine) on the linker backbone. The cleavage of the these functional groups cause a series of reactions, that eliminate the thiol trace and the remaining linker components. Such linkers could be used not only in detecting biomolecules but also in targeted drug deliveries.

[0027] AR may be a benzene unit or may comprises one or more N atoms in the aromatic ring.

[0028] Further, cleavable group CG may be according to formula IIIWith L selected from the group consisting ofwith R12-R17: independently H, D, F, Cl, Br, substituted / functionalized or unsubstituted alkyl groups. The dashed lines symbolize the connection points to the units Z and SP.

[0029] CG may be selected from units that are cleavable by treating the conjugates with reducing reagents.

[0030] The cleavage reaction can be carried out by treating with an appropriately selected reagent, such as phosphine (TCEP, THPP etc), as well as by thiol based reducing reagents such as DTT, BMS, DMPS etc. The linkers include one or more self-eliminating components adjacent to one or more cleavable groups.

[0031] Most preferred are self-eliminating cleavable conjugates according to one of the formulas A, B or C, wherein CG, AR, SP, Z, Y and Label are selected from the options discloedWith R= H, CH3.

[0032] The cleavable group can be substituted disulfide (-CR2-SS-CR2-), oxymethylene disulfide (-OCH(R)-SS-) where R = H, Me or azidomethine (-CHN3-) etc. Such group are cleaved off upon treatment with a cleave reagent, which initiates a cascade of reactions eliminating linker component leaving a native form of the DNA and protein probes.

[0033] Another object of the invention is therefore a process to cleave self-eliminating conjugates with the embodiments and variants disclosed wherein CG is cleaved by treating the conjugates with reducing reagents.

[0034] Figure 3 shows some of the traceless linkers where the disulfide or azido group act as cleavable group, and benzyl or hetero-aromatic benzyl groups which undergo self-eliminating by 1,6- elimination reaction. The hetero atom can be at any position of the aromatic ring in all cases. The cleave process and the subsequent self-elimination of compound A is shown in scheme 1 as an example.

[0035] Preferable, the reducing agent is selected from the group consisting ofDTBA p

[0036] In another embodiment of the invention, the self-eliminating cleavable conjugates may comprise a RM unit that is a nucleotide where the 3 ’OH is capped by a chemically removable group according to formula (IV), where B stands for a natural or artificial nucleobase.

[0037] Further variants and embodiments are disclosed as follows:

[0038] In one embodiment of this invention include uses of benzyl group attached to oxymethylene disulfide or its alkyl substituted analogues as shown in Fig. 3 (probe - carbamate - benzyl - oxymethylene - disulfide - spacer - fluorescent label). In this case, the disulfide group is the site of initial cleavage, which upon cleavage with reducing agents such as TCEP, THPP, DMPS, DTT or BMS etc initiates a cascade of further reactions. In the chain the benzyl group can be configured as para or meta position, and the aromatic ring can be with or without a substituting group. The benzyl group can be have heteroatoms (such as N). The groups Ri, R2, R3, and R4 can be independently H, D, methyl, ethyl, -SO3H, -CF3 or any alkyl or substituted alkyl group. The point of attachment to theprobes, shown here as X can be -NH as in carbamate, phosphate, sulfonate, or it could be -O- as well in carbonate. The substituent of the benzyl ring (R) can be one or multiple, and the ring can be configured para (A) or meta position (B), and the benzyl group can have hetero atom (C) as shown in the figure 4. The spacer can be PEG, peptide, carbohydrate or hydrocarbon / alkyl chain etc.

[0039] In another embodiment, the benzyl group can be attached to azidomethyl (-OCHN3-) as shown in figure 5 (probe - carbamate - benzyl - azidomethine - spacer - fluorescence dye label). The pending azido group can act as the trigger group. Phosphine based reagents such as TCEP, THPP can be used to cleave off the azide group and initiate the self-elimination process of the benzyl group via 1,6-elimination. In this case too, the benzyl group can be para or meta configuration and it can be substituted by one or more substituents groups (R = H, NO2, Cl, F, Br, Me, Et, CF3, - SO3H etc) and / or by hetero-atoms on the aromatic ring. The substituent Rl, R2, R3 and R4 can be H, D, Me, CF3, any alkyl or any substituted alkyl group.

[0040] In another embodiment, the invention includes the benzyl or thio-benzyl group attached to substituted ethyl disulfide group via a carbonate group (Figure 6). The substitution of the alpha carbons of the disulfide by alkyl groups provides extra stability to the former. The ethylene and the benzyl group undergo self-elimination only when the disulfide group is cleaved off. The benzyl carbonate and ethylene disulfide serve not only as spacer but also self-eliminating group. The substituent groups Ri, R2, R3, R4, Rs, Re, R etc can be independently H, D, NO2, Cl, F, Br, Me, Et, CF3, alkyl, substituted alkyl,- SO3H etc. The spacer (SP) can be PEG, alkyl chain, peptide, or carbohydrate chain.

[0041] Yet another embodiment of the invention includes use of the substituted ethylene group attached to double disulfide chain as shown in figure 7. The double disulfide chain serves as not only spacer, but also self-eliminating group through intra-molecular thiol interaction. Some of the examples double disulfide moiety can be BMS (bis(2-mercaptoethylsulfone), DTT (dithiothreitol), PDT (propyl dithio), 1,2-dimercapto ethyl or propyl etc (Figure 7). How the cleavage of the BMS linker can trigger intra-molecular attack to another disulfide for self-dissociation is shown in the scheme 2. The alpha carbon of the disulfide chain cab be substituted by one or more than one alkyl group (Ri-Re: independently H, D, Me, Et etc) to improve stability of the chain or to prevent non-specific interaction with thiol group in biological samples. Two specific example are shown in figure 7 (B) and 7(C).

[0042] Yet another embodiment includes benzyl-ethylene attached to double disulfide as shown figure 8). Again, in this case, cleavage of one of the disulfide can trigger a series of self-eliminating reaction leaving probes without trace of linkers. A specific example includes, benzyl is attached substituted ethyl and BMS figure 8 (B).

[0043] In all cases described above, the linker may be derivatized by functional group for coupling by “click chemistry” such as azide, acetylene, BCN, DMCO, TCO, DIFO, tetrazine, maleimide etc and the label groups can be fluorescent dyes, biotin, or any hapten groups.

[0044] The other aspects of this invention is ALL the linkers described herein can be attached to nucleoside, nucleotides or nucleic acids for application in new generation DNA sequencing (NGS). The nucleotides can have 3 ’-OH capped with reversible groups such as azido methyl (-CH2N3), substituted azidomethyl {-CHR-N3, R = -Me, H, -CH2F, -CH2-CH2F etc}, methylene disulfide {- CH(R)SSMe, R = H, Me, Et} etc (Fig 10). Due to self-elimination, they leave little or no trace or scars upon cleavage. Such linker can prevent the scars build up on the DNA chain in the sequencing cycle and could potentially help achieve longer read lengthy The cleavage mechanism reversibly terminating labeled nucleotide with self-eliminating linkers are shown in Scheme-3.EXAMPLES

[0045] To establish a proof of concept, NHS activated form of a model compound (6) was synthesis as described scheme -1, it was then conjugated to a nucleotide. Using the nucleotide conjugate (8), the stability, cleavage and self-immolation mechanism were studied by cleaving with DMPS and TCEP. We observed that the linker was stable - no detectable decomposition product was observed when heated for 40 mins at 70 degree C in Tris buffer pH 8.0 As expected, when the disulfide bond was cleaved off by DMPS or TCEP, the intermediate thio-hemiacetal and benzyl group fell off instantaneously, leaving no detectable intermediates (by UPLC). This absence of the intermediates, at detectable level by UPLC, confirmed a rapid self-elimination process of the linker upon cleavage.(i) TMSOTf, collidine, DCMAcetic anhydride -pyridine

[0046] Scheme -1: Synthesis of a model compound, in NHS activated form, compound (6)

[0047] Compound 6 was synthesized by a series of chemical reactions as shown in scheme 1.Steps are explained here:Synthesis of compound 2'.

[0048] 2.04 gm (8.56 mmol) dissolved in 16.0 mL DCM was added with 9.0 mL vinyl ethyl ether. It was then added with 115 mg pyridinium p-toluenesulfonate (PPTS) and stirred at RT for 1 h. The reaction mixture was concentrated by rotary evaporation, resuspended with 50 mL EtOAc and washed with 0.1 M NaHCCL (50 mL) and dried over Na2SO4. It was then purified by Flash Chromatography: the product was dissolved in ~ 15 mL hexane / EtOAc and purified by solid injection (80 gm HP Silica RediSepRf, Teledyne, flow rate: 60 mL / min, 0-5 min 100% hexane, then up to 20% EtOAc. Yield: 2.24 gm (84% yield). 'H-NMR (400 MHz, CDCh): 6 7.25 (2H, d), 6.95 (2H, d), 5.35 (1H, q), 4.68 (2H, s), 3.79 (1H, m), 3.54 (1H, m), 1.50 (3H, d), 1.21 (3H, t), 0.93 (9H, s) and 0.09 (6H, s) ppm.Synthesis of compound 3.

[0049] 2.22 gm of compound (2) dissolved in 25 mL DCM, cooled on an ice bath, added with collidine (2.8 mmL) followed by dropwise addition of TMSOTf (trimethylsilyltriflate) (2.6 mL) under argon gas. The mixture was stirred for 30 mins at ice-bath temperature. Ice bath was removed and 18-crown-6-ether (3.8 gm), and k-thiotosylate (3.24 gm) were added. It was stirred for Ih and brought to RT. then the thiol compound (1.91 mmol) added and stirred for Ih at RT. The reaction was then diluted with 100 mL EtOAc and washed with 0.1 M NaHCOs (2X 100 mL). The EtOAc part was then dried over Na2SO4, and concentrated by rotary evaporator. The product was then purified by silica gel column chromatography (80 gm HP Silica, Taledyne, flow 60 mL / min, 100% Hex for 5 mins, then 30% EtOAc over 30 mins. Yield: 0.46 gm (15.4% yield). 'H-NMR (400 MHz, CDCh): 8 7.27-7.29 (m, 3H), 6.97-7.00 (m, 2H), 5.38 (q, J = 6.2 Hz, IH), 4.71 (s, 2H), 3.77 (td, J = 7.0, 1.4 Hz, 2H), 1.85-1.80 (2H, m), 1.80-1.75 (3H, m), 1.32-1.27 (6H, m), 0.95 (s, 10H) and 0.09 (s, 7H) ppm.Synthesis of compound 4.

[0050] Compound (3) obtained from previous step (0.46 g, 1.1 mmol) was dissolved in anhydrous pyridine (3.0 mL) under Ar-atmosphere. Then acetic anhydride (0.13 mL, 1.3 mmol) was added, and stirred 24hr at room temperature. Then the reaction mixture was quenched by adding 1.0 mL MeOH. The product was then dissolved in -100 mL EtOAc and washed with water (2X100 mL). The EtOAc part was then washed with citric acid (1.0 M, lOOmL), followed by sodium bicarbonate solution (saturated, 100 mL). EtOAc part was then dried over Na2SO4, and purified by Flash Chromatography (40 gm, HP Teledyne), method: 0-3 min 100% hexane, then gradient up to 20% EtOAc over -20 mins, flow rate: 25 mL / min. Yield: 0.45 gm (80.2% yield). 'H-NMR (400 MHz, CHLOROFORM-D): 8 7.25 (d, 2H), 6.95 (d, 2H), 5.35 (IH, q), 4.68 (s, 2H), 4.15 (t, 2H), 2.08 (3H, s), 1.85 (t, 2H), 1.75 (3H, d), 1.25 (s, 6H), 0.95 (s, 9H) and 0.10 (6H, s) ppm.Synthesis of compound 5

[0051] 0.45 gm (0.98 mmol) of compound 4 was dissolved in 20 mL of dry THF under argon gas. The flask (100 mL) was then cooled under ice bath. It was added with 1.0 M TBAF / THF: 1.078 mL (1.1 eq). The mixture was then stirred on an ice water bath for 2 h. TLC showed complete conversion with product’s Rf= 0.3 / EtOAc:Hex(7:3). It was quenched with 100 mL brine and extracted with EtOAc (100X1 mL). The EtOAc part was dried over Na2SO4 and purified using 40 gm HP Teledyne column (100% Hex for 3 min, followed by 50% EtOAc over 25 mins. Target peak ~ 40% EtOAc. Yield: 0.2485 gm (76%). 'H-NMR (400 MHz, CDCh): 8 7.30 (2H, d), 6.96 (2H, d), 5.36 (1H, q), 4.63 (2H, s), 4.13 (2H, t), 2.02 (3H, s), 1.85 (2H, t), 1.74 (3H, 2 singlets, diastereotopic protons) and 1.27 (6H, s) ppm.Synthesis of compound 6.

[0052] 0.2436 gm of compound 5 (0.71 mmol) was dissolved in 5.0 mL dry acetonitrile. It was added with 236 mg DSC (1.3 eq, 0.923 mmol) and 161 uL of DIPEA (1.3 eq, 0.923 mmol) and stirred under Ar for overnight. It was then quenched by adding 100 mL EtOAc and 100 mL 1.0 M citric acid. It was partitioned and the EtOAc part was then washed again with saturated NaHCOv The EtOAc part was then dried over Na2SO4 and purified by flash chromatography using 40gm silica HP Teledyne column, 0 - 90% Hex-EtOAc as mobile phase. Yield: 115 mg ( 33%). 'H-NMR (CDCh): 7.35 (2H, d), 6.98 (2H, d), 5.37 (1H, q), 5.25 (2H, s), 4.16 (2H, t), 2.83 (4H, s), 2.04 (3H, s), 1.86 (2H, t). 1.75 (3H, two singlets, diasterotopic) and 1.25 (6H, s) ppm.

[0053] Compound 6, which is an activated form (in NHS state) was used to conjugate to PA- nucleotide to study stability of the linker and cleavage & self-immolation process as followsConjugation of compound 6 to amino modified reversible nucleotide terminator (7):

[0054] 4.0 umols of compound (7) in 2.0 mL of deionized water was added with 400 uL of 0.5M Na2HPO4. Then 2.0 eq (8.0 umols ~ 4.0 mg) activated linker (6) dissolved in -900 uL of DMF was added and stirred at room temperature for 1 h at room temperature. Conversion to the product 8 was confirmed by LC-MS. The product was purified by Flash chromatography using 40 gm C18-Aq column / Tel edyne ISCO with mobile phase 0 - 60% B over 30 mins, A = 100 mM TEAB, B = acetonitrile. The target fractions were combined and lyophilized. The product was dissolved in TE buffer, pH 8.5, and determined the concentration by UV-Vis spectroscopy, C = 0.7 mM, V = 2.0 mL, 1.5 urnol / - 40% yield, (according to scheme 2)

[0055] This product was used for studying stability, cleavage and self-immolation mechanism as follows: Stability: The linker conjugated compound (8) in IxTE buffer was very stable. When the compound 20 uL@ 0.7mM diluted in 100 uL of 10 mM Tris-HCl buffer, pH 8.0 was heated for 40 mins at 70 degree C, no detectable decomposition products were observed by UPLC.

[0056] Scheme-2: Synthesis of linker conjugated nucleotide reversible terminator (8)Cleavage and Self-Immolation:

[0057] 20 uL of 0.70 mM N3dUTP-Ben-Me-SS-Ac ( compound 8) was diluted in 100 uL deionized water. It was added with 20 uL of cleave reagent DMPS (40 mM) in 100 mM Tris-HCl buffer (final ~ 0.1 mM compound 8 and 5.7 mM DMPS). This mixture was heated at 70 degree C for 5 minutes. Then immediately analyzed by UPLC by injecting 10 uL of sample. We observed that the compound was fully cleaved off and only compound 11 was observed. The product was confirmed by LC-MS. No detectable amount of intermediate compounds, such as compound 9 and compound 10, were observed by UPLC. On the other hand, at room temperature compound 8 underwent very slow cleavage, producing compound 11 only about 20% after 25 minutes at room temperature (scheme-3).

[0058] Again, no intermediate compound 9 and 11 were observed. On the other hand, when the compound 8 (@ 0.2 mM final concentration) was treated with 15 mM TCEP in Tris-HCl buffer at pH 8.0, at room temperature, it cleaved off 100% the -SS-bond quantitatively converting to compound 11(with 10% reduction of the azido group (producing compound 12) in 3 mins, and -40% in 15 mins by UPLC), Scheme-3. Again no intermediate compounds 9 and 10 were observed by UPLC.

[0059] These results proved that the cleavable linker consisting of-[benzyl-O(CHMe)-SS- C(Me)2]- was relatively stable to thiol based reducing compound at room temperature as shown to DMPS, but could be cleaved off and undergo rapid self-immolation reaction at elevated temperature. On the other hand with more powerful reducing reagents such as with TCEP, the cleavage and self- immolation reaction could be achieved even at room temperature. The relative stability toward thiol based groups while cleavability by TCEP are important attributes of this type of cleavable linker for use in probing biological samples where native thiol groups are present abundantly.

[0060] Scheme-3: Cleavage and self-immolation mechanism of linker conjugated nucleotide reversible terminatorX = -NH, -O etc

[0061] Scheme 4: Cleavage and self- elimination / self-immolation mechanism leaving no trace of the linker components (trace / scars) on the antibody or DNA probes.ntibody H2N DNA

[0062] Scheme 5: Self-elimination mechanism of cleavable linker consisting of BMS chain and ethylene functional group.

[0063] Scheme 6: Cleavage of the self-eliminating linker and 3 ’capping group. The end product contains only hydroxymethyl group, which exist in natural nucleotides

[0064] Synthesis of self-immolating linker containing ethylene disulfide in activated forms (compounds 16A / B and 19A / B) as in scheme -7:

[0065] Scheme 7: Synthesis of ethylene disulfide based self-eliminating linker in NHS activated form for coupling to probes.Synthesis of compound 16A:

[0066] To a solution of Py-SS-EtOH (13A) (190.7 mg, 1.0 mmol, 1 eq.) in anhydrous MeOH (10 mL) in a 25 mL flask under Ar was added solid FmocNHEt(Me)2SH (14) (329.7 mg, 1.0 mmol, 1 eq.). The RXN mixture was stirred at room temperature overnight.

[0067] It was then concentrated under reduced pressure. The residue (yellow syrup) was purified by flash chromatography on ISCO Silica 24 g Gold; Gradient: EtOAc / hexane (0-100%), 35 mL / min. The product fractions were combined and concentrated under reduced pressure to give product 15A as a colorless syrup (392.1 mg, 96%). This compound treated with 1.5 eq of DSC-DIPEA in ACN overnight under argon gas, as in the synthesis of compound 6 (scheme - 1), to produce NHS activated compound 16ASynthesis of compound 16B:

[0068] To a solution of Py-SS-PrOH (13B) (251 mg, 1.25 mmol, 1 eq.) in anhydrous MeOH (12 mL) in a 25 mL RBF under Ar atmopshere was added solid FmocNHEt(Me)2SH (14) (408 mg, 1.0 mmol, 1 eq.).

[0069] The reaction mixture was stirred at room temperature over overnight. It was then concentrated under reduced pressure. The residue (yellow syrup) was purified by flash chromatography on ISCO Silica 24 g Gold; Gradient: EtOAc / hexane (0-100%), 35 mL / min. The product fractions were combined and concentrated under reduced pressure to give product 15B as a colorless syrup (506.7 mg, 97%). UPLC-MS QC: -95% pure UPLC(265 nm). This compound was activated to NHS form, asdescribed in the synthesis of compound 6 using DSC-DIPEA in CAN (scheme -1), to produce compound 16B.

[0070] Both compound 16A and 16B can be used as a linker to attach to DNA and antibody probes. The Fmoc group can be then deprotected by treat with 5% piperidine-water solution. The product can be used to label with NHS activated dye molecules to produce labeled probe.

[0071] On the other hand, compound 15A and 15B can be further reacted to install spacer (e.g. PEG) and activated to NHS form as shown in scheme-8, to produce 19A / B (scheme -8)

[0072] Scheme 8 : Synthesis of ethylene disulfide based self-eliminating linker with PEG spacer in NHS activated form for coupling to probes.Synthesis of NHS activated self-immolating linker 23:

[0073] Scheme 9: Synthesis of Self-eliminating linker with oxymethylene group in the NHS activated form for coupling to probes.Synthesis of compound 21A.

[0074] To a stirred mixture of 4-OH-Bn-OH (20) (501 mg, 4.0 mmol, 1.0 eq) and CS2CO3 (3.91 g, 12 mmol, 3.0 eq) in anhydrous DMF (10 mL) under Ar. It was added neat MTMC1 (0.5 mL, 6.0 mmol, 1.5 eq) slowly. The resulting RXN mixture was stirred at room temperature overnight. The completion of conversion was confirmed by UPLC-MS. The reaction mixture was quenched with water (10 mL, diluted with EtOAc (115 mL) and separated by partitioning. The organic layer was washed with water (5x10 mL), followed by a brine solution (2x10 mL), dried (over Na2SO4) and concentrated under reduced pressure. The residue was purified by flash chromatography on ISCO Silica 40 g Gold; Gradient: EtOAc / hexane (0-50%), 40 mL / min. The product fractions were combined and concentrated under reduced pressure to give product 21A as a colorless oil (502 mg, 67%).Synthesis of compound 21B

[0075] To a stirred mixture of 4-MTMO-Bn-OH 21A (502 mg, 2.72 mmol, 1.0 eq) in anhydrous pyridine (5 mL) under Ar was added AC2O (0.32 mL, 3.4 mmol, 1.25 eq). The resulting RXN mixture was stirred at room temperature overnight. The reaction mixture was quenched with MeOH (1 mL),diluted with EtOAc (100 mL) / water (10 mL) and separated. The organic layer was washed with citric acid (1.0 M in water, 3x10 mL), brine (2x10 mL), dried (Na2SO4) and concentrated under reduced pressure. The residue was purified by flash chromatography on ISCO Silica 24 g Gold; Gradient: EtOAc / hexane (0-50%), 35 mL / min. The product fractions were combined and concentrated under reduced pressure to give product 21B as a colorless oil (562.8 mg, 91.3%).JH NMR (400 MHz, CDCh) = 8 7.33 (d, J= 8.8 Hz, 2H), 6.97 (d, J= 8.8 Hz, 2H), 5.17 (s, 2H), 5.07 (s, 2H), 2.27 (s, 3H), 2.10 (s, 3H).Synthesis of compound 22'.

[0076] To a stirred solution of 4-MTMO-Bn-OAc 21B (560 mg, 2.47 mmol, 1.0 eq.) in DCM (2.5 mL, anhydrous) under Ar at room temperature, was added SO2CI2 (1.00 M in DCM, 3.09 mL, 3.09 mmol, 1.25 eq ). The resulting reaction mixture was stirred for 1 hour. The completion of the conversion was confirmed by UPLC-MS (SM to 4-0 H. reactive intermediate -OCH2CI hydrolyzed on UPLC column). The mixture was then concentrated under reduced pressure to give a pale yellow syrup. It was dissolved in DCM / DMF (anhydrous 2.572.5 mL) under Ar. TsSK solid (650 mg, 2.87 mmol, 1.5 eq.) was then added in one portion. The resulting reaction mixture was stirred at room temperature for -1 hour at room temperature. UPLC clearly indicated a full conversion. Then FmocNHEt(Me)2SH solid (1.22 g, 3.71 mmol, 1.5 eq ) was added to the mixture in one portion. The resulting reaction mixture was stirred at room temperature under Ar overnight.

[0077] The reaction mixture was diluted with EtOAc (100 mL) and washed with water (5x10 mL), NaHCOs sat., 2xl0mL), brine (2x10 mL), dried (Na2SO4) and concentrated under reduced pressure. The residue was purified by flash chromatography on ISCO Silica 40 g Gold; Gradient: EtOAc / hexane (0~100%), 40 mL / min. The product fractions were combined and concentrated under reduced pressure to give product 22 as a colorless oil (1.59 g). The material was repurified by flash chromatography on ISCO Silica 80 g Gold; Gradient: EtOAc / hexane (0~75%), 60 mL / min to give product 22 as a colorless oil (1.46 g) >80% pure by UPLC). 'H NMR (400 MHz, CDCh): 8 7.79 (d, J = 7.3 Hz, 2H), 7.62 (d, J= 7.3 Hz, 2H), 7.42 (t, J= 7.3 Hz, 2H), 7.33 (m, 4H), 6.94 (d, J= 8.6 Hz, 2H), 5.35 (t, J= 6.4 Hz, 1H, NH), 5.32 (s, 2H), 5.06 (s, 2H), 4.44 (d, J= 6.8 Hz, 2H), 4.25 (t, J= 6.8 Hz, 1H), 3.28 (d, J= 6.4 Hz, 2H), 2.10 (s, 3H) and 1.30 (s, 9H) ppm.

[0078] Compound 22 is a key compound to install spacer such as PEG. This compound when treated with NaOH-EtOH solution, both the Ac- and Fmoc protecting groups get removed. Then NHS-PEG-Fmoc reaction in DIPEA-DMF followed by activation of the benzyl OH with DSC-DIPEA in ACN produced the compound 23. Using compound 23, DNA and antibody probes can be labeledaccording to scheme 10. Also it be converted -Ns derivative and label to DNA and antibody probes functionalized with “clickable” counterpart (e.g. alkyne, BCN, DIFO, TCO, DMCO, tetrazene etc) as in scheme -11.

[0079] Scheme 10: Labeling of probes with self-eliminating linkersCGD = Click Chem functions group (terminal alkyne, BNC, tetrazene etc)= Fluorescent dye

[0080] Scheme 11 : Synthesis of “click chemistry” compatible linker and labeling to probes by click chemistry.

[0081] An example of self-immolating cleavable linker consisting of a cleavable group azido (CG = -CHN3-), in NHS activated form, is compound 34. It can be synthesized according to Scheme - 12.

[0082] A catalytic amount of PPTS helps the amino functionalized vinyl compound (29B) to react with the phenolic -OH group compound 29, as in Scheme 1, to produce an ethyl acetal compound 30.

[0083] The latter (30) can be then activated with SnCh and react with TMSN3 to produce an azido compound -31. Alternatively, it can be created by activating with TMSOTf- collidine in DCM, followed by treating with NaN?. The latter can treated with NH4OH to remove the PhCO group protection to produce compound 32. It can be linked to PEG spacer after DSC activation.

[0084] Then deprotection of the TBDMS by treating with TBAF / THF and then labeling the amino terminal with NHS activated dye, followed by activation of the benzyl hydroxyl group product compound 34. This compound can be then used to label any probe, such as nucleotide, antibody or DNA with amino functional group. Not only that, it can be converted to “clickable” functional group (such as terminal alkyne, BCN, tetrazene, TCO, DMCO etc).

[0085] Scheme 12: Synthesis of NHS activated self-eliminating linker with {benzyl - OCH(Ns)- and PEG spacer labeled with dye.

Claims

CLAIMS1. Self-eliminating cleavable conjugates according to general formula I or IIWithCG: cleavable group comprising at least one residue selected from the group consisting ofwith Re-Rii: independently H, D, F, Cl, Br, substituted or unsubstituted alkyl groups having 1 to 10 caron atoms and Me = CH3.AR: aromatic 6 membered ringRM: recognizing moiety selected from the group consisting of DNA, antibody, nucleotideRi to Rs: independently H, alkyl with 1 to 10 carbon atoms, Cl, F, -OR, -SR, -NR2 or - NHR with R = alkyl having 1 to 10 carbon atomsSP: spacer unit selected from the group consisting of PEG, peptide chain or alkyl chain having 2 to 10 carbon atomsZ: -O- or -S-Y : linking functional group comprising at least one selected from the groups consisting of carbonate : -O(C=O)O-, carbamate: -NH(C=O)O-, thio-carbonate: -S(C=O)O-, phosphate: - OP(=O)(OH)O- or sulfonate: -OS(=O)2-O-.Label: fluorescent unit2. Self-eliminating cleavable conjugates according to claim 1 characterized in that AR comprises one or more N atoms in the aromatic ring.

3. Self-eliminating cleavable conjugates according to claim 1 or 2 characterized in that CG is according to formula IIIWith L selected from the group consisting ofwith R12-R17: independently H, D, F, Cl, Br, substituted / functionalized or unsubstituted alkyl groups.

4. Self-eliminating cleavable conjugates according to any of claims 1 to 3 characterized in that RM is a nucleotide where the 3 ’OH is capped by a chemically removable group according to formula (IV), where B stands for a nucleobaseR-i = -H, D, -Me, -Et, -CH2F, -5. Self-eliminating cleavable conjugates according to any of claims 1 to 4 characterized in that CG, AR, SP, Z and Y are selected to define the self-eliminating cleavable conjugates according to one of the formulas A, B or CWith R= H, CH3.

6. Process to cleave self-eliminating conjugates according to any of claims 1 to 5 characterized in that CG is cleaved by treating the conjugates with reducing reagents.

7. Process according to claim 6 characterized in that the reducing agent is selected from the group consisting ofDTBA p8. Self-eliminating cleavable conjugates according to any of claims 1 to 6 characterized in that the label is selected from the group consisting of fluorescence dyes, biotin, polypeptide, haptens, nitrobenzyl, steroids and carbohydrates.

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