Solid-phase “one-step” Synthesis of Peptide-Drug Conjugates (PDCs) based on Tumor-targeting Peptide/Cell-penetrating Peptide and SN38

doi:10.6023/A26010030

Abstract

Tumors, particularly drug-resistant malignant tumors, pose a severe threat to human health, underscoring the importance of developing novel antitumor therapeutics. 7-Ethyl-10-hydroxycamptothecin (SN38), a representative derivative of camptothecin (CPT), is a highly potent chemotherapeutic agent that exhibits broad-spectrum antitumor activity. Nevertheless, the clinical application of SN38 has been significantly limited by its poor water solubility, short half-life, and low tumor selectivity. Peptide-drug conjugates (PDCs) represent an emerging class of targeted antitumor therapeutics, offering distinct advantages including strong tumor tissue penetration, low immunogenicity and ease of synthesis and structural optimization. Conjugating SN38 with peptides could markedly improve its water solubility, tumor targeting capability, and antitumor potential. Conventional synthesis of peptide-SN38 based conjugates typically relies on a “two-step” strategy, involving solid-phase peptide synthesis (SPPS) to prepare the peptide segment, followed by solution-phase construction of peptide-SN38 conjugates. This approach is cumbersome, often leading to poor yields and a time-consuming workflow. In this study, we developed a streamlined solid-phase “one-step” synthesis strategy, wherein both peptide assembly and SN38 conjugation were performed sequentially on the solid support. The target peptide-SN38 conjugates could be readily obtained through direct peptide cleavage and purification. Utilizing different tumor-targeting peptides (LHRH and Octreotide) and cell-penetrating peptide (CPP12), three distinct series of peptide-SN38 conjugates were designed and efficiently synthesized. We systematically investigated the effects of different conjugation linkers and cleavage conditions on the synthetic efficiency, establishing the straightforward solid-phase “one-step” synthesis strategy. Ultimately, three conjugate series, LHRH-SN38, octreotide-SN38, and CPP12-SN38 were efficiently synthesized, yielding eight structurally well-defined peptide-SN38 conjugates. In vitro cytotoxicity assays indicated that both LHRH-SN38 and octreotide-SN38 conjugates exhibit potent antitumor activity, significantly enhancing SN38's selectivity toward tumor cells while reducing its toxicity to normal cells. Solubility and cellular uptake experiments demonstrated that all three series of peptide-SN38 conjugates substantially improved the aqueous solubility of SN38 and significantly enhanced SN38 uptake by tumor cells. Furthermore, wound-healing assays indicated that conjugation of SN38 to three kinds of peptides notably enhanced its inhibitory effect on tumor cell migration. By employing the “one-step” solid-phase synthesis strategy, this study systematically investigated the synthetic approaches for peptide-SN38 conjugates derived from diverse peptide architectures, including linear peptide (LHRH), disulfide-bridged cyclic peptide (Octreotide), and amide-bridged cyclic peptide (CPP12). This study not only provides the robust and efficient synthetic strategy for PDCs based on SN38 and other CPT-derived chemotherapeutics, but also offers valuable insights for the development of novel antitumor therapeutics.

Key words: CPP12, LHRH, octreotide, peptide-drug conjugates (PDCs), peptide synthesis, SN38, solid-phase "one-step" synthesis strategy, tumor-targeting peptide

Cite this article

Changbo Fang, Zi Xu, Shuya Zhu, Ming Meng, Bin Zhang, Yunkun Qi, Shanshan Du. Solid-phase “one-step” Synthesis of Peptide-Drug Conjugates (PDCs) based on Tumor-targeting Peptide/Cell-penetrating Peptide and SN38[J]. Acta Chimica Sinica, 2026, 84(6): 916-932.

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Fig. & Tab. 17

Figure 1. (A) The structures of camptothecin and its derivatives; (B) Structures of pH-dependent lactone and carboxylate forms of SN38; (C) Method for the synthesis of Boc-SN38-COOH

Figure 2. (A) PDCs constructed by conjugating SN38 with LHRH, Octreotide, and CPP12; (B) Physical and chemical property improvements of SN38 after conjugation with the polypeptide

Figure 3. (A) Two-step synthesis of peptide-SN38 conjugates; (B) One-step synthesis of peptide-SN38 conjugates

Figure 4. (A) The solid phase peptide synthesis process for LS-1a~LS-3c; (B) The structures of LS-1a~LS-3c

Figure 5. Analytical RP-HPLC and ESI-MS spectra of the (A) LS-1a*; (B) LS-1b*; (C) LS-1c*; (E) LS-2a; (F) LS-2b; (G) LS-2c*; (I) LS-3a; (J) LS-3b; (K) LS-3c. Analytical RP-HPLC spectra for the co-injection of (D) LS-1a* and LS-1b*; (H) LS-2a and LS-2b; (L) LS-3a, LS-3b and LS-3c

Peptides		Peptide sequence	Peptide cleavage cocktails	ESI-MS		RP-HPLC purity^a/%
Peptides		Peptide sequence	Peptide cleavage cocktails	Calculated mass/Da	Observed mass/Da	RP-HPLC purity^a/%
LS-1	LS-1a*	pGlu-HWSYk(-AEEA-SN38)LRPG-NH₂	TFA/TIPS/H₂O	1873.090	1499.484	—
	LS-1b*		TFA/TIPS	1873.090	1498.146	—
	LS-1c*		TFA/H₂O	1873.090	—	—
LS-2	LS-2a	pGlu-HWSYk(-rrrr-SN38)LRPG-NH₂	TFA/TIPS/H₂O	2352.659	2352.505	32.4
	LS-2b		TFA/TIPS	2352.659	2352.454	26.7
	LS-2c*		TFA/H₂O	2352.659	—	—
LS-3	LS-3a	pGlu-HWSYk(-SN38)LRPG-NH₂	TFA/TIPS/H₂O	1727.903	1727.679	39.3
	LS-3b		TFA/TIPS	1727.903	1727.803	46.9
	LS-3c		TFA/H₂O	1727.903	1727.412	26.9

Table 1. The peptide sequence, peptide cleavage conditions, molecular weight and RP-HPLC purity for peptide LS-1~LS-3

Figure 6. (A) The solid phase peptide synthesis process for OS-1a~OS-4b; (B) The structures of OS-1a~OS-4b

Figure 7. Analytical RP-HPLC and ESI-MS spectra of the (A) OS-1a; (B) OS-1b; (D) OS-2a; (E) OS-2b; (G) OS-3a; (H) OS-3b*; (J) OS-4a; (K) OS-4b. Analytical RP-HPLC spectra for the co-injection of (C) OS-1a and OS-1b; (F) OS-2a and OS-2b; (I) OS-3a; (L) OS-4a and OS-4b

Peptides		Peptide sequence	Peptide cleavage cocktails	ESI-MS		RP-HPLC purity^a/%
Peptides		Peptide sequence	Peptide cleavage cocktails	Calculated mass/Da	Observed mass/Da	RP-HPLC purity^a/%
OS-1	OS-1a		TFA/TIPS/H₂O	1669.398	1668.610	52.1
OS-1	OS-1b		TFA/TIPS	1669.398	1668.656	52.7
OS-2	OS-2a		TFA/TIPS/H₂O	2148.454	2147.980	50.2
OS-2	OS-2b		TFA/TIPS	2148.454	2148.236	26.5
OS-3	OS-3a		TFA/TIPS/H₂O	1523.698	1523.304	37.2
OS-3	OS-3b*		TFA/TIPS	1523.698	—	—
OS-4	OS-4a		TFA/TIPS/H₂O	1693.910	1693.574	31.7
OS-4	OS-4b		TFA/TIPS	1693.910	1693.768	30.0

Table 2. The peptide sequence, peptide cleavage condition, molecular weight and RP-HPLC purity for peptide OS-1~OS-4

Figure 8. (A) The solid phase peptide synthesis of CS-1 and CS-2; (B) The structures of CS-1 and CS-2

Figure 9. Analytical RP-HPLC and ESI-MS spectra of the (A) CS-1 and (C) CS-2. Analytical RP-HPLC spectra of (B) purified CS-1 and (D) purified CS-2

Peptides	Peptide sequence	Peptide cleavage cocktails	ESI-MS		RP-HPLC purity^a/%
Peptides	Peptide sequence	Peptide cleavage cocktails	Calculated mass/Da	Observed mass/Da	RP-HPLC purity^a/%
CS-1		TFA/TIPS/H₂O	1992.280	1992.184	39.4
CS-2		TFA/TIPS/H₂O	1847.122	1846.877	38.2

Table 3. The peptide sequence, peptide cleavage conditions, molecular weight and RP-HPLC purity for peptide CS-1 and CS-2

	OS-1	OS-2	OS-3	OS-4	LS-2	LS-3	CS-1	CS-2	SN38
MDA-MB-231	6.1±0.5	3.7±0.3	10.1±0.9	8.9±0.9	2.7±0.2	4.7±0.4	3.6±0.3	5.4±0.6	<0.3
MCF-7	1.2±0.1	0.3±0.1	6.3±0.7	3.6±0.2	0.5±0.1	1.9±0.2	3.1±0.3	4.7±0.5	<0.3
HepG2	2.6±0.2	1.3±0.2	5.1±0.5	4.1±0.4	4.3±0.6	6.5±0.6	3.9±0.3	4.6±0.6	<0.3
HUVEC	64.4±4.6	26.3±2.2	>100	>100	26.9±2.7	73.7±5.3	4.0±0.4	5.4±0.5	<0.3
L929	40.6±3.7	20.8±2.3	>100	>100	16.6±1.2	47.1±4.0	4.9±0.3	6.7±0.7	<0.3
SI^a	53.7	87.7	—	—	53.8	38.8	1.3	1.1	—
SI^b	33.8	69.3	—	—	33.2	24.8	1.6	1.4	—

Table 4. IC50 values for SN38 and its peptide conjugates after 48 h of incubation

Figure 10. (A) UV-vis spectrum of SN38. Standard curves for (B) LS-2; (C) OS-2; (D) CS-1; (E) CS-2; (F) SN38 at different concentrations. λ=365 nm, n=3, mean±SEM. (G) and (H) Water solubility of SN38 and its peptide conjugates. n=3, mean±SEM, ****P<0.0001

Figure 11. (A) Fluorescence images of MCF-7 cells treated with OS-1, OS-2, OS-3 and OS-4. (B) Fluorescence images of MCF-7 cells treated with LS-2 and LS-3. (C) Fluorescence images of MCF-7 cells treated with CS-1 and CS-2. (D) Fluorescence images of MCF-7 cells treated with SN38. (E) Quantification of uptake ratios for OS-1, OS-2, OS-3, OS-4 and SN38. n=3, mean±SD. (F) Quantification of uptake ratios for LS-2, LS-3 and SN38. n=3, mean±SD. (G) Quantification of uptake ratios for CS-1, CS-2 and SN38. n=3, mean±SD. ns: not significant, *P<0.05, **P<0.01, ***P<0.001

Figure 12. Anti-migration effects of SN38, CS-1, CS-2, LS-2 and OS-2 against HepG2 cells. (A) Migration images of MCF-7 cells at 0, 12, and 24 h. (B) Quantification of wound healing rate of MCF-7 cells at 12 h and 24 h. n=3, mean±SD. (C) Migration images of HepG2 cells at 0, 12, and 24 h. (D) Quantification of wound healing rate of HepG2 cells at 12 h and 24 h. n=3, mean±SD. *P<0.05, **P<0.01, ***P<0.001

Figure 13. Serum stability assay. Analytical RP-HPLC spectra of the hydrolysis of (A) CS-1, (B) OS-2 and (C) LS-2 by human serum; (D) Human serum stability of CS-1, OS-2 and LS-2

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固相“一步法”合成基于肿瘤靶向肽/细胞穿透肽和SN38的多肽-药物偶联物(PDCs)