Name: Mal-amido-PEG4-acid | Mal-amido-PEG4-acid
Brand: Ambeed
SKU: A748445

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Synonyms: Mal-amido-PEG4-acid

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Quality Control of Mal-amido-PEG4-acid Purity: 98% SDS Specification

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Product Details of Mal-amido-PEG4-acid

CAS No. :	1263045-16-4
Formula :	C₁₈H₂₈N₂O₉
M.W :	416.42
SMILES Code :	OC(CCOCCOCCOCCOCCNC(CCN1C(C=CC1=O)=O)=O)=O
Synonyms :	Mal-amido-PEG4-acid
MDL No. :	MFCD13184954
InChI Key :	IAJVEYLYYHOZEY-UHFFFAOYSA-N
Pubchem ID :	51340949

Safety of Mal-amido-PEG4-acid

GHS Pictogram:
Signal Word:	Warning
Hazard Statements:	H315-H319
Precautionary Statements:	P264-P280-P302+P352-P337+P313-P305+P351+P338-P362+P364-P332+P313

Application In Synthesis of Mal-amido-PEG4-acid

* All experimental methods are cited from the reference, please refer to the original source for details. We do not guarantee the accuracy of the content in the reference.

Downstream synthetic route of [ 1263045-16-4 ]

[ 1263045-16-4 ] Synthesis Path-Downstream 1~30

1
[ 1343407-40-8 ]
[ 1263045-16-4 ]
[ 1343407-44-2 ]

Yield	Reaction Conditions	Operation in experiment
50%	With triethylamine; In dichloromethane; N,N-dimethyl-formamide; at 20℃;	Amine 10 (99 mg, 0.109 mmol, 1 eq) was added to a solution of NHS-PEG4-Maleimide (Thermo Scientific, 61.6 mg, 0.120 mmol, 1.1 eq) and TEA (18.2 μL, 0.130 mmol, 1.2 eq) in a mixture of anhydrous DCM (5 mL) and DMF (1 mL). The reaction was allowed to stir at room temperature overnight at which point it was found to be almost complete by LC/MS (3.27 min (ES+) m/z (relative intensity) 1307.55 ([M+H]+., 100)). The volatiles were removed by evaporation under reduced pressure. The residue was purified by flash chromatography (gradient from 3/97 up to 5/95 methanol/chloroform) to yield 71 mg (50%) of pure product 13.[0440]Analytical Data: 1H NMR (400 MHz, CDCl3) δ 8.58 (s, 1H), 7.50 (d, 2H, J=8.47 Hz), 7.28 (s, 1H), 7.25-7.20 (m, 2H), 7.18-7.01 (m, 9H), 6.89 (d, 1H, J=7.58 Hz), 6.79 (d, 2H, J=8.68 Hz), 6.59 (s, 2H), 6.51 (s, 1H), 5.77 (d, 1H, J=6.42 Hz), 5.25 (d, 1H, J=11.43 Hz), 4.83-4.64 (m, 2H), 4.63-4.49 (m, 1H), 4.43-4.38 (m, 1H), 4.18 (s, 1H), 3.96-3.85 (m, 1H), 3.84 (s, 3H), 3.76-3.56 (m, 9H), 3.57-3.34 (m, 15H), 3.34-3.20 (m, 3H), 3.15 (dd, 1H, J=14.22 Hz, J=5.60 Hz), 3.07-2.89 (m, 4H), 2.48-2.29 (m, 4H), 1.97-1.90 (m, 1H), 1.61-1.39 (m, 3H), 1.35 (s, 9H), 1.29-1.12 (m, 4H). MS (ES+) m/z (relative intensity) 1307.55 ([M+H]+., 100).

References: [1]Patent: US2013/35484,2013,A1 .Location in patent: Paragraph 0438; 0439; 0440.

2
[ 756525-91-4 ]
[ 1263045-16-4 ]

References: [1]Patent: WO2014/100762,2014,A1 .

3
[ 55750-62-4 ]
[ 1615234-93-9 ]
[ 1263045-16-4 ]

Yield	Reaction Conditions	Operation in experiment
	With N-ethyl-N,N-diisopropylamine; In dichloromethane; at 20℃;	Referring to the scheme of synthesis of Compound O, β-alanine was treated with maleic anhydride in DMF and the acid so obtained was reacted with N-hydroxysuccinimide (NHS) under DCC coupling to give NHS-ester. The BOC protective group in commercially available t-blc-N-amido-dPEG4-acid was removed by treatment with TFA to give the TFA salt of the amine, which was reacted with previously synthesized NHS ester. The carboxylic acid so obtained was isolated and was coupled with N-hydroxysuccinimide using EDCI to furnish NHS ester Compound O.
0.227g	With N-ethyl-N,N-diisopropylamine; In dichloromethane; at 20℃;	[0277] Referring to the scheme of synthesis of Compound O, β-alanine was treated with maleic anhydride in DMF and the acid so obtained was reacted with N-hydroxysuccinimide (NHS) under DCC coupling to give NHS-ester. The BOC protective group in commercially available t-boc-N-amido-dPEG4-acid was removed by treatment with TFA to give the TFA salt of the amine, which was reacted with previously synthesized NHS ester. The carboxylic acid so obtained was isolated and was coupled with N-hydroxysuccinimide using EDCI to furnish NHS ester Compound O.

References: [1]Patent: WO2014/100762,2014,A1 .Location in patent: Paragraph 240; 248.
[2]Patent: WO2015/196167,2015,A1 .Location in patent: Paragraph 0269; 0277.

4
[ 6066-82-6 ]
[ 1263045-16-4 ]
[ 756525-99-2 ]

Yield	Reaction Conditions	Operation in experiment
0.109 g	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride;	Referring to the scheme of synthesis of Compound O, β-alanine was treated with maleic anhydride in DMF and the acid so obtained was reacted with N-hydroxysuccinimide (NHS) under DCC coupling to give NHS-ester. The BOC protective group in commercially available t-blc-N-amido-dPEG4-acid was removed by treatment with TFA to give the TFA salt of the amine, which was reacted with previously synthesized NHS ester. The carboxylic acid so obtained was isolated and was coupled with N-hydroxysuccinimide using EDCI to furnish NHS ester Compound O.
0.109 g	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride;	Synthesis of linker-drugReferring to the scheme of synthesis of Compound O, β-alanine was treated with maleic anhydride in DMF and the acid so obtained was reacted with N-hydroxysuccinimide (NHS) under DCC coupling to give NHS-ester. The BOC protective group in commercially available t-boc-N-amido-dPEG4-acid was removed by treatment with TFA to give the TFA salt of the amine, which was reacted with previously synthesized NHS ester. The carboxylic acid so obtained was isolated and was coupled with N-hydroxysuccinimide using EDCI to furnish NHS ester Compound O.
0.109g	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride;	[0277] Referring to the scheme of synthesis of Compound O, β-alanine was treated with maleic anhydride in DMF and the acid so obtained was reacted with N-hydroxysuccinimide (NHS) under DCC coupling to give NHS-ester. The BOC protective group in commercially available t-boc-N-amido-dPEG4-acid was removed by treatment with TFA to give the TFA salt of the amine, which was reacted with previously synthesized NHS ester. The carboxylic acid so obtained was isolated and was coupled with N-hydroxysuccinimide using EDCI to furnish NHS ester Compound O.

330 mg

With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃; for 2h;

The product 4-11 (1.0 g, 2.4 mmol) obtained in the previous step and EDC-HCl (0.55 g, 2.88 mmol) were dissolved in 25 mL DCM, HOSu (0.33 g, 2.88 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. 50 mL water was added, the mixture was then extracted with EtOAc for 3 times (50 mL×3). The organic phases were combined, washed with saturated brine for 3 times (50 mL×3), dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by prep-HPLC to give 330 mg product as colorless oil, yield 27%. LCMS (ESI) m/z 514.2 (M+H)+.

References: [1]Patent: WO2014/100762,2014,A1 .Location in patent: Paragraph 240; 248.
[2]Patent: WO2015/196089,2015,A1 .Location in patent: Paragraph 0273; 0281.
[3]Patent: WO2015/196167,2015,A1 .Location in patent: Paragraph 0269; 0277.
[4]Patent: US10449258,2019,B2 .Location in patent: Page/Page column 133; 134; 137; 138.

5
[ 1263045-16-4 ]
(11S,11aS,11'S,11a'S)-di-tert-butyl 8,8'-(((5-(3-aminoprop-1-yn-1-yl)-1,3-phenylene)bis(methylene))bis(oxy))bis(7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate) [ No CAS ]
(11S,11aS,11'S,11a'S)-di-tert-butyl 8,8′-(((5-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,19-dioxo-7,10,13,16-tetraoxa-4,20-diazatricos-22-yn-23-yl)-1,3-phenylene)bis(methylene))bis(oxy))bis(7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate) [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
67 mg	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃; for 3h;Inert atmosphere;	(b) (11S,11aS,11'S,11a'S)-di-tert-butyl 8,8'-(((5-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,19-dioxo-7,10,13,16-tetraoxa-4,20-diazatricos-22-yn-23-yl)-1,3-phenylene)bis(methylene))bis(oxy))bis(7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate) (9) <strong>[1263045-16-4]MAL-dPEG4-acid</strong> (88 mg, 0.21 mmol) was added to a stirred solution of EDCI (41 mg, 0.21 mmol) and the crude primary amine 8 in dry DCM (4 mL) at room temperature. The reaction mixture was stirred under an argon atmosphere for 3 hours at which point analysis by LC/MS showed a substantial amount of desired product at retention time 3.58 min (ES+) m/z 1475 ([M+H]+, ˜10% relative intensity), 1498 ([M+Na]+, ˜5% relative intensity) accompanied by a side product at retention time 3.85 min. The reaction mixture was diluted with DCM (30 mL) and washed with H2O (3*10 mL), brine (20 mL), dried (MgSO4), filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (gradient elution: 100% DCM to 96:4 v/v DCM/MeOH) gave the maleimide 9 as a foam (67 mg, 22% yield over 2 steps).
67 mg	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃; for 3h;Inert atmosphere;	<strong>[1263045-16-4]MAL-dPEG4-acid</strong> (88 mg, 0.21 mmol) was added to a stirred solution of EDCI (41 mg, 0.21 mmol) and the crude primary amine 8 in dry DCM (4 mL) at room temperature. The reaction mixture was stirred under an argon atmosphere for 3 hours at which point analysis by LC/MS showed a substantial amount of desired product at retention time 3.58 min (ES+) mlz 1475 ([M+ H]+ , -10% relative intensity), 1498 ([M+ Na]+', -5% relative intensity) accompanied by a side product at retention time 3.85 min. The reaction mixture was diluted with DCM (30 mL) and washed with H20 (3 x 10 mL), brine (20 mL), dried (MgS04), filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (gradient elution: 100% DCM to 96:4 v/v DCM/MeOH) gave the maleimide 9 as a foam (67 m 22% yield over 2 steps).

References: [1]Patent: US2014/294868,2014,A1 .Location in patent: Paragraph 0610; 0611.
[2]Patent: WO2016/44396,2016,A1 .Location in patent: Paragraph 00381.

6
[ 1263045-16-4 ]
(11aS,11a'S)-8,8'-(((5-(4-(3-aminopropyl)piperazin-1-yl)-1,3-phenylene)bis(methylene))bis(oxy))bis(7-methoxy-2-methylene-2,3-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-5(11aH)-one) [ No CAS ]
N-(3-(4-(3,5-bis((((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)methyl)phenyl)piperazin-1-yl)propyl)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3,6,9,12-tetraoxapentadecan-15-amide [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
46 mg	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃; for 3h;Inert atmosphere;	(c) N-(3-(4-(3,5-bis((((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)methyl)phenyl)piperazin-1-yl)propyl)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3,6,9,12-tetraoxapentadecan-15-amide (13) <strong>[1263045-16-4]MAL-dPEG4-acid</strong> (42 mg, 0.10 mmol) was added to a stirred solution of EDCI (20 mg, 0.10 mmol) and the crude primary amine 12 (77 mg, 0.10 mmol) in dry DCM (4 mL) at room temperature. The reaction mixture was stirred under an argon atmosphere for 3 hours at which point analysis by LC/MS showed complete consumption of starting material, a substantial amount of desired product at retention time 2.42 min (ES+) m/z 1176 ([M+H2O]+, ˜5% relative intensity) and excess <strong>[1263045-16-4]MAL-dPEG4-acid</strong> at retention time 2.05 min (weak signal on diode array but detectable on ES+/ES-). The reaction mixture was diluted with DCM (30 mL) and washed with H2O (15 mL), brine (20 mL), dried (MgSO4), filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (gradient elution: 100% CHCl3 to 93:7 v/v CHCl3/MeOH) gave the maleimide 13 as a foam (46 mg, 55%). Note that trace amounts of excess <strong>[1263045-16-4]MAL-dPEG4-acid</strong> could not be removed using flash chromatography.

References: [1]Patent: US2014/294868,2014,A1 .Location in patent: Paragraph 0618; 0619.

7
[ 1263045-16-4 ]
(11aS,11a'S)-8,8'-(((5-(piperazin-1-yl)-1,3-phenylene)bis(methylene))bis(oxy))bis(7-methoxy-2-methylene-2,3-dihydro-1H-pyrrolo[2,1-c][1,4]-benzodiazepin-5(11aH)-one) [ No CAS ]
N-(15-(4-(3,5-bis((((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)methyl)phenyl)piperazin-1-yl)-15-oxo-3,6,9,12-tetraoxapentadecyl)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamide [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
32%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃; for 2h;Inert atmosphere;	(c) N-(15-(4-(3,5-bis((((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)methyl)phenyl)piperazin-1-yl)-15-oxo-3,6,9,12-tetraoxapentadecyl)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamide (17) N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (35 mg, 0.18 mmol, 1.1 eq) was added to a solution of compound (15) (116 mg, 0.165 mmol, 1.0 eq) and <strong>[1263045-16-4]1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16-tetraoxa-4-azanonadecan-19-oic acid</strong> (16) (69 mg, 0.165 mmol, 1.0 eq) in anhydrous DCM (5 mL) under Argon. The resultant solution was stirred at room temperature for 2 h. The reaction mixture was diluted with DCM (50 mL), washed with water (100 mL), saturated sodium hydrogen carbonate solution (100 mL), water (100 mL), brine (100 mL), dried (MgSO4) and evaporated under reduced pressure. Purification by flash column chromatography [CHCl3/MeOH 0% to 5% in 1% increments] gave the product 17 as a yellow glass (0.058 g, 32%) Analytical Data: [α]18D=[+628] (c=0.25, CHCl3); RT 2.65 min; MS (ES+) m/z (relative intensity) 1101 ([M+1]+, 40)

References: [1]Patent: US2014/294868,2014,A1 .Location in patent: Paragraph 0625-0628.
[2]Journal of Medicinal Chemistry,2017,vol. 60,p. 9490 - 9507.

8
[ 55750-62-4 ]
[ 756525-91-4 ]
[ 1263045-16-4 ]

Yield	Reaction Conditions	Operation in experiment
0.227 g		Synthesis of linker-drugReferring to the scheme of synthesis of Compound O, β-alanine was treated with maleic anhydride in DMF and the acid so obtained was reacted with N-hydroxysuccinimide (NHS) under DCC coupling to give NHS-ester. The BOC protective group in commercially available t-boc-N-amido-dPEG4-acid was removed by treatment with TFA to give the TFA salt of the amine, which was reacted with previously synthesized NHS ester. The carboxylic acid so obtained was isolated and was coupled with N-hydroxysuccinimide using EDCI to furnish NHS ester Compound O.

References: [1]Patent: WO2015/196089,2015,A1 .Location in patent: Paragraph 0273; 0281.

9
[ 7423-55-4 ]
[ 1263045-16-4 ]

References: [1]Patent: WO2015/196167,2015,A1 .
[2]Patent: US10449258,2019,B2 .

10
(R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-sulfopropanoic acid [ No CAS ]
[ 260367-12-2 ]
[ 1263045-16-4 ]
(R)-28-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-7,10,26-trioxo-8-(sulfomethyl)-3,13,16,19,22-pentaoxa-6,9,25-triazaoctacosan-1-oic acid [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
		The title compound was synthesized using solid phase peptide synthesis as described herein. 2-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)ethoxy)acetic acid (1543 mg) was dissolved in 10 mL dioxane, and the solvent was concentrated under reduced pressure. (The procedure was repeated twice). The material was lyophilized overnight. The dioxane-dried amino acid was dissolved in 20 mL sieve-dried dichloromethane to which was added N,N- diisopropylethylamine (4.07 mL). The solution was added to a 2-chlorotrityl solid support resin (8000 mg), which was previously washed (twice) with sieve-dried dichloromethane. The mixture of resin and amino acid was shaken at ambient temperature for 4 hours, drained, washed with 17:2: 1 dichloromethane:methanol:N,N-diisopropylethylamine, and washed three times with N,N- dimethylformamide. The mixture was then washed three more times, alternating between sieve-dried dichloromethane and methanol. The loaded resin was dried in a vacuum oven at 40 C. The resin loading was determined by quantitative Fmoc-loading test measuring absorbance at 301 nm of a solution obtained by deprotecting a known amount of resin by treatment with 20% piperidine in N,N- dimethylformamide. All Fmoc deprotection steps were performed by treatment of the resin with 20% piperidine in N,N-dimethylformamide for 20 minutes followed by a washing step with N,N- dimethylformamide. Coupling of the amino acids (R)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-3-sulfopropanoic acid and subsequently l-(2,5-dioxo-2,5-dihydro-lH- pyrrol-l-yl)-3 -oxo-7, 10, 13, 16-tetraoxa-4-azanonadecan-19-oic acid was done by activation of 4 equivalents of amino acid with 4 equivalents of ((lH-benzo[d][l,2,3]triazol-l-yl)oxy)tri(pyrrolidin-l- yl)phosphonium hexafluorophosphate(V) and 8 equivalents of N,N-diidopropylethylamine in N,N- dimethylformamide for one minute followed by incubation with the resin for one hour. The title compound was cleaved from the resin by treatment with 5 % trifluoroacetic acid in dichloromethane for 30 minutes. The resin was filtered, and the filtrate was concentrated under reduced pressure to yield the title compound which was used in the next step without further purification. MS (ESI) m/e 669.0 (M+H)+
		The title compound was synthesized using solid phase peptide synthesis as described herein. 2-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)ethoxy)acetic acid (1543 mg) was dissolved in 10 mLdioxane, and the solvent was concentrated under reduced pressure. (The procedure was repeated twice). The material was lyophilized overnight. The dioxane-dried amino acid was dissolved in 20 mL sieve-dried dichloromethane to which was added N,N-diisopropylethylamine (4.07 mL). The solution was added to a 2-chlorotrityl solid support resin (8000 mg), which was previously washed(twice) with sieve-dried dichloromethane. The mixture of resin and amino acid was shaken at ambient temperature for 4 hours, drained, washed with 17:2:1 dichloromethane:methanol:N,Ndiisopropylethylamine, and washed three times with N,N-dimethylformamide. The mixture was then washed three more times, alternating between sieve-dried dichloromethane and methanol. The loaded resin was dried in a vacuum oven at 40 C. The resin loading was determined by quantitative Fmoc20 loading test measuring absorbance at 301 nm of a solution obtained by deprotecting a known amountof resin by treatment with 20% piperidine in N,N-dimethylformamide. All Fmoc deprotection steps were performed by treatment of the resin with 20% piperidine in N,N-dimethylformamide for 20 minutes followed by a washing step with N,N-dimethylformamide. Coupling of the amino acids (R)2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-sulfopropanoic acid and subsequently 1 -(2,5-dioxo-2,5-dihydro- 1 H-pyrrol- 1 -yl)-3 -oxo-7, 10,13,1 6-tetraoxa-4-azanonadecan- 19-oic acid was done by activation of 4 equivalents of amino acid with 4 equivalents of ((1H-benzo[d][1,2,3]triazol-1- yl)oxy)tri(pyrrolidin- 1 -yl)phosphonium hexafluorophosphate(V) and 8 equivalents of N,Ndiidopropylethylamine in N,N-dimethylformamide for one minute followed by incubation with the resin for one hour. The title compound was cleaved from the resin by treatment with 5 %trifluoroacetic acid in dichloromethane for 30 minutes. The resin was filtered, and the filtrate was concentrated under reduced pressure to yield the title compound which was used in the next step without further purification. MS (ESI) m/e 669.0 (M+H).
		The title compound was synthesized using solid phase peptide synthesis as described herein. 2-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)ethoxy)acetic acid (1543 mg) was dissolved in 10 mL dioxane, and the solvent was concentrated under reduced pressure. (The procedure was repeated twice). The material was lyophilized overnight. The dioxane-dried amino acid was dissolved in 20 mL sieve-dried dichloromethane to which was added N,N-diisopropylethylamine (4.07 mL). The solution was added to a 2-chlorotrityl solid support resin (8000 mg), which was previously washed (twice) with sieve-dried dichloromethane. The mixture of resin and amino acid was shaken at ambient temperature for 4 hours, drained, washed with 17:2:1 dichloromethane:methanol:N,N-diisopropylethylamine, and washed three times with N,N-dimethylformamide. The mixture was then washed three more times, alternating between sieve-dried dichloromethane and methanol. The loaded resin was dried in a vacuum oven at 40 C. The resin loading was determined by quantitative Fmoc-loading test measuring absorbance at 301 nm of a solution obtained by deprotecting a known amount of resin by treatment with 20% piperidine in N,N-dimethylformamide. All Fmoc deprotection steps were performed by treatment of the resin with 20% piperidine in N,N-dimethylformamide for 20 minutes followed by a washing step with N,N-dimethylformamide. Coupling of the amino acids (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-sulfopropanoic acid and subsequently <strong>[1263045-16-4]1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16-tetraoxa-4-azanonadecan-19-oic acid</strong> was done by activation of 4 equivalents of amino acid with 4 equivalents of ((1H-benzo[d][1,2,3]triazol-1-yl)oxy)tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate(V) and 8 equivalents of N,N-diidopropylethylamine in N,N-dimethylformamide for one minute followed by incubation with the resin for one hour. The title compound was cleaved from the resin by treatment with 5% trifluoroacetic acid in dichloromethane for 30 minutes. The resin was filtered, and the filtrate was concentrated under reduced pressure to yield the title compound which was used in the next step without further purification. MS (ESI) m/e 669.0 (M+H)+.

References: [1]Patent: WO2016/94505,2016,A1 .Location in patent: Paragraph 000564.
[2]Patent: WO2017/214456,2017,A1 .Location in patent: Page/Page column 331.
[3]Patent: US2019/153107,2019,A1 .Location in patent: Paragraph 1043.

11
[ 1263045-16-4 ]
(11S,11aS,11'S,11a'S)-di-tert-butyl 8,8'-(((5-(3-aminopropa-1-yne-1-yl)-1,3 phenylene)bis(methylene))bis(oxy))bis(7-methoxy-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-10(5H)carboxylate) [ No CAS ]
(11S,11aS,11'S,11a'S)-di-tert-butyl 8,8'-(((5-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,19-dioxo-7,10,13,16-tetraoxa-4,20-diazatricos-22-yn-23-yl)-1,3-phenylene)bis(methylene))bis(oxy))bis(7-methoxy-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-10(5H)carboxylate) [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
80 mg	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃; for 3h;Inert atmosphere;	MAL-dPEG (registered trademark) 4-acid (37 mg, 0.089mmol) which, added to the DCM (4 ml) anhyride soln. stirring at room temperature in a second (17 mg, 0.089mmol) EDCI and roughness of a tertiary amine 1 8. The reaction mixture is stirred for 3 hours in an argon atmosphere, at this point of time by the analysis by LC/MS (method C), holding time by 1.69 (ES+) m/z 1450.55 ([M+H] +., about 10% relative intensity), 1498 ([M+Na] +., relative strength of about 80%) by, substantial amounts of the desired product is shown. The reaction mixture is diluted with DCM (30 ml), H 2 O (3×10mL), saline (20 ml) by washing, dehydrating (MgSO 4), by filtration, the crude product is evaporated in a vacuum. flash chromatography purified (gradient elution: from 100% DCM 96:4 vol./ vol. DCM/MeOH) by, a bubble obtd. alkylmaleimide 9 (80 mg, 2 at step 62% yield).

References: [1]Patent: JP2016/510806,2016,A .Location in patent: Paragraph 0399.

12
[ 1263045-16-4 ]
(11aS,11a'S)-8,8'-(((5-(piperazin-1-yl)-1,3-phenylene)bis(methylene))bis(oxy))bis(7-methoxy 2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)one) [ No CAS ]
N-(15-(4-(3,5-bis((((S)-7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)phenyl)piperazin-1-yl)-15-oxo-3,6,9,12-tetraoxapentadecyl)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamide [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
4.7 mg	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃; for 3h;Inert atmosphere;	MAL-dPEG (registered trademark) 4-acid (35 mg, 0.084mmol) which, added to the DCM (3 ml) anhyride soln. stirring at room temperature in a first (16 mg, 0.084mmol) EDCI and roughness of a tertiary amine 1 12. The reaction mixture is stirred for 3 hours in an argon atmosphere, at this point of time by the analysis by LC/MS (method C), dividing the holding time 1.24 (ES+) m/z 1077.40 ([M+H] +., 90% relative intensity) by, a substantial amount of the desired product is shown. The reaction mixture is diluted with DCM (30 ml), H 2 O (3×10mL), saline (20 ml) by washing, dehydrating (MgSO 4), by filtration, the crude product is evaporated in a vacuum. Preparative UPLC purified (gradient elution: 11 min. 87:13 vol./ vol. H 2 O/CH 3 CN over from 15:75 vol./ vol. H 2 O/CH 3 CN) by, a final product 13, obtained as a thin brown oil (4.7 mg, 5% yield at the step 2).

References: [1]Patent: JP2016/510806,2016,A .Location in patent: Paragraph 0403.

13
[ 1020412-43-4 ]
[ 1263045-16-4 ]
N-((4-amino-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-2-yl)methyl)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-ethyl-3,6,9,12-tetraoxapentadecan-15-amide [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
35%		A solution of MAL-PEG4-acid (265.7 mg, 0.638 mmol) and N,N’dicyclohexylcarbodiimide (DCC, 144.8 mg, 0.702 mmol) in dry dichloromethane / acetonitrile (1:1,5 mL) was stirred at room temperature for lh, followed by addition of compound 1(100mg, 0.319 mmol) in one portion. After 72h of stirring, volatile organic s were removed under vacuum. The residue obtained was purified by flash column chromatography on silica gel, eluting with step gradients of methanol in dichloromethane at a ratio of v/v 1:20, 1:15, and 1:9, to afford the target product N-((4-amino- 1 -(2-hydroxy-2-methylpropyl)- 1 H-imidazo [4,5-cj quinolin-2- yl)methyl)- 1 -(3-(2,5-dioxo-2,5-dthydro- 1H-pyrrol- 1 -yl)propanamido)-N-ethyl-3 ,6,9, 12- tetraoxapentadecan-15-amide (80 mg, 35% yield) as white colored foamy solid oil. ‘H NMR (300 MHz, CDC13) ö 8.40-7.82 (br m, 1H), 7.74 (d, 1=8.1 Hz, 1H), 7.44 (t, 1=7.5 Hz, 1H), 7.30 (t, 1=7.4 Hz, 1H), 6.76 - 6.28 (br m, 2H), 4.82 - 4.32 (br m, 2H), 4.08 - 3.64 (br m, 6H), 3.54 (brs, 14H), 3.31 (br s, 3H), 2.63 (br s, 2H), 2.38 (t, 1=6.9 Hz, 2H), 1.27 (br s, 4H), 1.20-0.68 (br m, 5H). MS (ESI+) mlz 712 (M+1), 734 (M+Na).

References: [1]Patent: WO2018/140831,2018,A2 .Location in patent: Paragraph 0627; 0628.

14
[ 1263045-16-4 ]
C₄₂H₇₂N₆O₈ [ No CAS ]
C₆₀H₉₈N₈O₁₆ [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
89%	With N-ethyl-N,N-diisopropylamine; In dichloromethane; at 20℃; for 72h;	β-Ala-MMAE (15.1 mg, 19.2 μmol) was dissolvedin DCM (0.5 mL). DIPEA (16.4 μL, 95.8 μmol) and mal-dPEG(4)-NHS (19.7 mg, 38.4 μmol) were added, and the reaction mixture wasstirred for 3 d at room temperature. The solvent was removed underreduced pressure and the crude product was purified by automatedcolumn chromatography (24 g column, DCM/MeOH) to afford the desiredproduct (20.3 mg, 89%). NMR spectrum is shown in Fig. S9. MS(ESI-TOF): calcd. for C60H98N8O16 1209.6993 [M+Na]+, 616.3443[M+2Na]2+; found 1209.6967 [M+Na]+, 616.3435 [M+2Na]2+.

References: [1]Journal of Controlled Release,2019,vol. 300,p. 13 - 21.

15
[ 68858-20-8 ]
[ 1263045-16-4 ]
[ 71989-26-9 ]
N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-oyl]-L-valyl-N₆-(tert-butoxycarbonyl)-L-lysine [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
46%		2-chlorotrityl resin (1.6 mmol/g, 2 grams) was added to reaction vessel, and washed with DMF 2 times. The resin was swelled in 20 mL DMF for 10 minutes, and then drained. Fmoc- Fys(Boc)-OH (937 mg, 2mmol) and DIPEA (0.7 mL, 4 mmol) dissolved in 10 mL DMF was added to the resin and shake for 30 minutes at room temperature. MeOH (5 mL) was added to the resin and shaken for 5 min, then drained, and washed with DMF 5 times. The substitution was assumed to be 1 mmol/g. The resin washed with DCM 3 times, washed with MeOH 3 times, then dried under high vacuum overnight. The prepared Fmoc-Fys(Boc)-2-chlorotrityl resin (1 gram) was added to a reaction vessel. The resin washed with DMF 3 times and swelled in 10 mL DMF for 10 minutes, then drained. The Fmoc was deprotected using the general deprotected procedure. Using the general coupling procedure Fmoc-Val-OH was coupling to the resin, followed by the general deprotection procedure. MP-PEG4-OH was coupled using the general coupling procedure. The resin was then washed with DCM 3 times, followed by MeOH 3 times, and placed under high vacuum overnight. The peptide was cleaved off resin by stirring the resin in a solution of 1 mL Acetic Acid, 2 mL hexaflouroisopropanol, and 7 mL DCM for 1 hour. Resin was then filtered and rinsed with DCM 3 times, and then the solution was concentrated in vacuo. The white powder was dissolved in 2:1 DMA:H20 (3 mL) and purified by preparative HPFC using a 30 x 250 mm Phenomenex Max-RP 4 pm Synergi 80A reverse phase column using a 5-60-95% gradient elution of MeCN (0.05% TFA) in aqueous 0.05% TFA described below. Fractions containing the desired product were lyophilized to afford a white powder (343 mg, 0.461 mmol, 46%). Rt = 1.50 min General Method UPLC. MS (m/z) [M + H]+ calc for C34H57N5O13 744.16, found 744.40. 5-60-95% Gradient Elution

References: [1]Patent: WO2019/195665,2019,A1 .Location in patent: Paragraph 0337; 0342; 0343.

16
[ 68858-20-8 ]
[ 1263045-16-4 ]
[ 71989-26-9 ]
[ 56-40-6 ]
MP-PEG₄-VK(Boc)G-OH [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
40%		Unprotected glycine pre-loaded 1.1 mmol/g on 2-chlorotryityl resin was purchased from BAChem. Resin (1 gram) was added to reaction vessel. Resin washed with DMF 4 times and drained completely. Resin swelled by shaking in DMF for 30 minutes, and drained. Using the general coupling procedure Fmoc-Lys(Boc)-OH was coupled to the resin. The Fmoc was deprotected using the general deprotection procedure. Using the general coupling procedure Fmoc-Val-OH was coupled to the resin, followed by the general deprotection procedure. MP- PEG4-OH was coupled using the general coupling procedure. The resin was then washed with DCM 3 times, followed by MeOH 3 times, and placed under high vacuum overnight. The peptide was cleaved from the resin by stirring the resin in a solution of 1 mL Acetic Acid, 2 mL hexaflouroisopropanol, and 7 mL DCM for 1 hour. Resin was then filtered and rinsed with DCM (0763) 3 times, and then the solution was concentrated in vacuo. The white powder was dissolved in 2:1 DMA:H20 (3 mL) and purified by preparative HPLC using a 30 x 250 mm Phenomenex Max-RP (0764) 4 pm Synergi 80A reverse phase column using a 5-60-95% gradient elution of MeCN (0.05% TFA) in aqueous 0.05% TFA described below. Fractions containing the desired product were lyophilized to afford a white powder (354 mg, 0.442 mmol, 40%). Rt = 1.39 min General Method UPLC. MS (m/z) [M + H]+ calc for C36H59N6O14 801.42, found 801.02.5-60-95% Gradient Elution General Fmoc deprotection procedure (0768) [0342] A solution of 20% piperidine in DMF (10 mL) was added to the resin, shaken for 1 minute, and drained. Another 10 mL of 20% piperidine in DMF was added to the resin, shaken for 30 minutes, and drained. The resin washed with DMF 4 times and drained completely. General Coupling Procedure (0769) [0343] A solution was prepared in DMF (10 mL) of Fmoc Amino Acid (3 mmol), HATU (3 mmol), DIPEA (6 mmol). The solution was added to the resin, and shaken for 60 minutes. The reaction vessel was drained and washed with DMF 4 times.

References: [1]Patent: WO2019/195665,2019,A1 .Location in patent: Paragraph 0341; 0342; 0343.

17
[ 1263045-16-4 ]
MP-PEG₄-Val-Lys-7-MAD-MDCPT [ No CAS ]