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Bioconjugation Linkers, Crosslinkers & Spacers

A practical library of NHS-ester, maleimide/thiol, click (DBCO↔Azide, TCO↔Tetrazine), glycan/aldehyde, photo-reactive linkers and PEG spacers—including cleavable vs non-cleavable and enzymatic, site-specific systems. Built for ADC/ODC design, labeling and nanoparticle interfaces.

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Overview

Bio-Synthesis designs and manufactures a diverse range of linkers, spacers, and crosslinkers that enable precise and reproducible bioconjugation across biomolecules such as antibodies (ADCs), oligonucleotides (ODCs), peptides, proteins, enzymes, small molecules, lipids, carbohydrates, and nanomaterials. By optimizing the reactive group, spacer length, and release mechanism, our reagents preserve biomolecular activity and ensure stable, high-yield conjugate formation.

The performance of any bioconjugation reagent depends on its reactive chemistry, which governs how it covalently attaches to a target molecule. Each linker or crosslinker is engineered to react selectively with functional groups naturally present in proteins, peptides, or nucleic acids to produce efficient, site-specific conjugation.

Labeling reagents typically contain a single reactive group—such as an N-hydroxysuccinimide (NHS) ester for amine coupling—paired with a functional tag like a fluorescent dye, biotin, or affinity ligand. Crosslinkers, in contrast, feature two or more reactive groups, forming durable molecular bridges between biomolecules for stable and defined conjugates.

At Bio-Synthesis, we offer a comprehensive portfolio of bioconjugation chemistries and custom linker development services, including amine-reactive, thiol-reactive, carbonyl-reactive, and bioorthogonal click-chemistry linkers. Our catalog of linkers and spacers supports applications in antibody labeling, enzyme immobilization, drug conjugation, nucleic acid tagging, and biosensor design.

By selecting the appropriate reactive chemistry—targeting primary amines, thiols, carbonyls, azides, or alkynes—Bio-Synthesis helps researchers achieve high specificity, efficient coupling, and robust conjugate stability for reliable protein, nucleic acid, and small-molecule bioconjugation workflows.

ISO 9001:2015 / ISO 13485:2016 45+ Years of Expertise U.S. Facilities – Texas Bench to Kilo Scale Production Confidential & IP-Protected

Service at a Glance

  • Custom linkers, spacers, and crosslinkers for bioconjugation
  • Amine, thiol, carbonyl, and click-chemistry coupling strategies
  • Cleavable and non-cleavable linker for ADC, ODC development
  • PEG, alkyl, and heterobifunctional spacer engineering
  • Comprehensive QC: LC-MS, HPLC, UV/Vis, and activity assays
  • Feasibility studies within days. Confidential & IP-protected.

Why Bio-Synthesis

  • 45+ years of experience in linker and conjugation chemistry
  • ISO 9001:2015 / 13485:2016 certified U.S. facilities
  • Integrated peptide, oligo, and conjugation manufacturing in-house
  • Bench-to-multi-gram scale with full analytical QC support
  • Expert consultation for research, diagnostic, and therapeutic projects
  • US, Texas locations. NDAs/MSAs available upon request.

Linkers & Spacers for Bioconjugation

NHS ester reacts with primary amine to form a stable amide conjugate
Reagent / Example Functional Group Notes / Applications
DSS, BS³, Sulfo‑NHS esters; PEG‑NHS; BIS‑PEG‑NHS Succinimidyl ester (NHS) Fast Lys/N‑terminus acylation (pH 7.2–8.0); water‑soluble options reduce aggregation.
PEG‑PFP ester; Bis‑PEG‑PFP Pentafluorophenyl ester (PFP) More hydrophobic leaving group; efficient amine coupling in mixed aqueous/organic media.
PEG‑acid; Bis‑PEG‑acid Carboxylic acid (–COOH) Activate in situ (EDC/NHS or DIC/NHS) to form NHS esters for amine coupling; great for surfaces/polymers.
PEG‑PNP carbonate; mPEG‑PNP carbonate p‑Nitrophenyl carbonate (PNP) Forms carbamates with amines; useful for PEGylation and installing stable urethane linkages.
FITC and other –N=C=S dyes; aryl isocyanates Isothiocyanate / Isocyanate Classic protein labeling; verify F/P and spectral compatibility.
Bromo‑PEG, Chloro‑PEG, Iodo‑PEG Alkyl halide (–CH₂–X) SN2 alkylation of amines under basic, less‑selective conditions; best treated as building blocks.
PEG‑Tosylate (PEG‑OTs) p‑Toluenesulfonate (OTs) Good leaving group for downstream substitution; not amine‑selective in aqueous buffers.
Product Category Typical Labels Notes
PEG NHS ester mPEG‑NHS, PEG‑NHS Primary workhorse for Lys/N‑terminus coupling.
BIS‑PEG‑NHS α,ω‑bis‑NHS‑PEG Crosslinking or surface bridging via two NHS ends.
PEG PFP ester PEG‑PFP Hydrophobic activated ester; good in mixed solvents.
PEG PNP carbonate PEG‑PNP carbonate Amine→carbamate installs (urethane linkages).
PEG acid HOOC‑PEG‑R Activate with EDC/NHS or DIC/NHS in situ.
Bis‑PEG‑acid HOOC‑PEG‑(…)-COOH Scaffold for further activation/multi‑point coupling.
Bromo PEG / Chloro PEG / Iodo PEG PEG‑Br / PEG‑Cl / PEG‑I Building blocks for SN2 derivatization (less selective for amines).
PEG Tosylate PEG‑OTs Versatile leaving group for synthesis (non‑selective bioconj.).
Non‑PEG linker LC / aryl spacers Compact or rigid spacing when PEG is not desired.
PEG aldehyde* PEG‑CHO *Carbonyl‑reactive (for aminooxy/hydrazide); included for cross‑panel completeness.
Technical Notes
  • Use PBS/HEPES pH 7.2–7.5; avoid Tris/glycine that consume NHS-esters.
  • LC/PEG spacers reduce steric clash on dense surfaces.
Sample Submission
  • Protein ≥0.5–1.0 mg at ≥1 mg/mL (≥95% purity); provide buffer and target F/P.
Recommended Reading
  1. Mattson, G., et al. (1993). A practical approach to crosslinking. Molecular Biology Reports 17:167–183.
  2. Grabarek, Z., and Gergely, J. (1990). Zero-length crosslinking procedure with the use of active esters. Analytical Biochemistry 185:131–135.
  3. Staros, J.V., et al. (1986). Enhancement by N-hydroxysulfosuccinimide of water‑soluble carbodiimide‑mediated coupling reactions. Analytical Biochemistry 156:220–222.
  4. Timkovich, R. (1977). Detection of the stable addition of carbodiimide to proteins. Analytical Biochemistry 79:135–143.

maleimide Thiol reaction form stable thioether bond
Reagent / Example Functional Group Notes / Applications
SMCC / Sulfo-SMCC, MBS; Mal-PEG-NHS; Mal-PEG-Azide Maleimide Fast, selective Cys coupling (pH ~6.5–7.5) → stable thioether; add 1–2 mM EDTA. Optional ring-opening (post-coupling) improves long-term stability.
Iodoacetyl-PEG (IA-PEG); Bromoacetyl-PEG (BA-PEG); Chloroacetyl-PEG (CA-PEG) Haloacetyl Irreversible S-alkylation; reactivity I > Br >> Cl. Light-sensitive; higher pH increases off-target amine reaction—optimize at pH 7.5–8.3.
SPDP-PEG / OPSS-PEG / PDP-PEG; SPDP, LC-SPDP, SATA Pyridyl disulfide Disulfide exchange gives reversible S–S link; monitor 2-thiopyridone at 343 nm. Useful for triggered intracellular release (reductive cytosol).
VS-PEG-NHS; VS-PEG-Azide; heterobifunctional VS-PEG-(X) Vinyl sulfone Michael addition to thiolate (pH ~7–8.5); slower than maleimide but forms very stable thioether; tolerant of amines.
Epoxy-PEG (glycidyl-PEG); epoxy-activated surfaces Epoxide (glycidyl) Less selective—reacts with thiols/amines/hydroxyls at pH ≥8.5; good for polymer/surface coupling when broad reactivity is acceptable.
PEG-Br / PEG-Cl / PEG-I; PEG-Tosylate (PEG-OTs) Halide / Tosylate (leaving groups) Used to build thiol-reactive PEGs by SN2 in organic media; not thiol-selective in aqueous bioconjugation. List under “Building Blocks”.
Category Example Product Names Functional Group Typical Formats Primary Use
Maleimide Linkers Mal-PEGn-NHS; Mal-PEGn-Azide; Mal-PEGn-DBCO; SMCC; Sulfo-SMCC; MBS Maleimide PEG2/4/8/12/24; linear/branched; heterobifunctional Fast, selective Cys coupling (near-neutral pH) → stable thioether
Iodo PEG IA-PEGn-NHS; IA-PEGn-COOH Iodoacetyl PEG4–PEG24; heterobifunctional Irreversible S-alkylation; highest haloacetyl reactivity
Bromo PEG BA-PEGn-NHS; BA-PEGn-COOH Bromoacetyl PEG4–PEG24; heterobifunctional Robust S-alkylation; slightly slower than iodoacetyl
Chloro PEG CA-PEGn-NHS; CA-PEGn-COOH Chloroacetyl PEG4–PEG24; heterobifunctional Slower haloacetyl; typically used at slightly higher pH
SPDP PEG SPDP-PEGn; OPSS-PEGn; PDP-PEGn Pyridyl disulfide PEG4–PEG24; heterobifunctional Cleavable disulfide exchange; 2-thiopyridone release at 343 nm
PEG Tosylate* PEG-OTs (tosylate) Leaving group Discrete PEGn or polymer PEG MWs Building block to synthesize thiol-reactive PEGs; not selective in aqueous bioconjugation

*Use PEG-OTs to prepare maleimide-PEG or haloacetyl-PEG via SN2 in organic media before biomolecule coupling.

Technical Notes
  • Remove thiols (DTT/TCEP) before maleimide steps; add 1–2 mM EDTA.
  • For re-bridging, use bis-maleimide after mild partial reduction.
  • Buffer: include 1–2 mM EDTA (prevents metal-catalyzed thiol oxidation).
  • Maleimide selectivity drops above pH ~8.3 (amine addition); couple near neutral.
  • Vinyl sulfone is slower than maleimide but gives highly stable adducts.
  • Haloacetyl reagents are more reactive but less chemoselective at higher pH; protect from light.
  • Epoxides are broad-reactivity tools—prefer surfaces/polymers rather than precise protein site-modification.
Sample Submission
  • Antibody ≥1 mg at ≥1 mg/mL; specify desired DAR range; ship 2–8 °C.
Recommended Reading
  1. Ishikawa, E., et al. (1983). Enzyme-labeling of antibodies. J Immunoassay 4:209–327.
  2. Brinkley, M.A. (1992). A survey of methods for preparing protein conjugates with dyes, haptens and cross-linking reagents. Bioconjugate Chem 3:2–13.
  3. Hashida, S., et al. (1984). More useful maleimide compounds for the conjugation of Fab to horseradish peroxidase through thiol groups in the hinge. J Appl Biochem 6:56–63.
  4. Mattson, G., et al. (1993). A practical approach to cross-linking. Molecular Biology Reports 17:167–83.
  5. Partis, M.D., et al. (1983). Cross-linking of proteins by omega-maleimido alkanoyl N-hydroxysuccinimide esters. J Protein Chem 2:263–77.
  6. Yoshitake, S., et al. (1982). Mild and efficient conjugation of rabbit Fab and horseradish peroxidase using a maleimide compound and its use for enzyme immunoassay. J Biochem 92:1413–24.

azide modified biomolecule react with DBCO to form copper-free click reaction
Reaction Handles Notes / Applications
CuAAC Azide + Alkyne + Cu(I) Quantitative; avoid copper for live systems (use SPAAC/IEDDA).
SPAAC DBCO/BCN + Azide Copper-free; ideal for live-cell, in vivo and nanoparticles.
IEDDA TCO + Tetrazine Ultra-fast; supports orthogonal dual payloads.
Category Common Formats / Handles Representative Products Notes
Alkyne & Azide Alkyne (–C≡CH), Azide (–N₃); NHS-activated, maleimide, amine, COOH; phosphoramidites (oligos) Alkyne-PEGn-NHS; Azide-PEGn-NHS; Azide-PEGn-maleimide; 5′-Azide oligo phosphoramidites For CuAAC or SPAAC; the most versatile way to install click handles.
DBCO (SPAAC) DBCO-PEGn-NHS; DBCO-PEGn-maleimide; DBCO-amine DBCO-PEG4/8/12/24-NHS; DBCO-PEGn-Maleimide Copper-free reaction with azides; fast, protein-friendly.
BCN (SPAAC) BCN-PEGn-NHS; BCN-maleimide; BCN-amine BCN-PEGn-NHS; BCN-PEGn-Maleimide Compact SPAAC partner; excellent on proteins & surfaces.
TCO (IEDDA) TCO-PEGn-NHS; TCO-maleimide; TCO-amine TCO-PEGn-NHS; TCO-PEGn-Maleimide Ultra-fast with tetrazines; ideal for live-cell/in vivo work.
Tetrazine (IEDDA) Tetrazine-PEGn-NHS; Tetrazine-maleimide; Tetrazine-amine Tetrazine-PEGn-NHS; Me-Tz / Ph-Tz variants Pairs with TCO; tune electronics for speed vs stability.
Click Reaction Ligands (CuAAC) Cu(I)-stabilizing ligands; additives TBTA, THPTA, BTTAA, BTTP; sodium ascorbate kits Accelerate CuAAC and mitigate copper-induced damage.
Technical Notes
  • Pair SPAAC (DBCO↔Azide) with IEDDA (TCO↔Tetrazine) for dual systems.
  • Prefer SPAAC/IEDDA for live cells; ensure no azide in CuAAC workflows.
Sample Submission
  • Provide azide/alkyne or TCO/tetrazine handles on at least one partner.
  • Oligos: 5–10 nmol (ss) or ≥20 nmol (ds) with end-handles.
Recommended Reading
  1. Agard, N., et al. (2006). A comparative study of bioorthogonal reactions with azides. ACS Chemical Biology 1(10):644–648.
  2. Prescher, J.A. and Bertozzi, C.R. (2005). Chemistry in living systems. Nature Chemical Biology 1(1):13–21.
  3. Varki, A., et al. (2008). Essentials of Glycobiology, 2nd ed. Cold Spring Harbor Press: Cold Spring Harbor, NY.
  4. Saxon, E. and Bertozzi, C. (2000). Cell surface engineering by a modified Staudinger reaction. Science 287:2007–2010.
  5. Berlett, B. and Stadtman, E. (1997). Protein oxidation in aging, disease, and oxidative stress. Journal of Biological Chemistry 272(33):20313–20316.
  6. Nessen, M.A., et al. (2009). Selective enrichment of azide-containing peptides from complex mixtures. Journal of Proteome Research 8(7):3702–3711.

diazirine reacts cross link with biomolecule through UV radiation
Reagent Activation Notes / Applications
Diazirine UV (≈350–365 nm) Short-lived carbene; efficient protein crosslinking; widely used in live-cell mapping.
Benzophenone UV (≈365 nm) Triplet biradical abstracts H•; reusable upon further irradiation; good for membranes/surfaces.
Aryl azide (general) UV (≈254–365 nm) Nitrene insertion into C–H / N–H bonds; broad photoaffinity labeling.
Psoralen UVA (≈320–365 nm) Intercalates DNA/RNA; forms thymine crosslinks; nucleic-acid photoaffinity and mapping.
Phenyl azide UV (≈254–365 nm) Parent aryl-azide; generates nitrenes for general photo-crosslinking.
o-Hydroxyphenyl azide (o-HPA) UV (≈350–365 nm) Can form dehydroazepines; reacts readily with nucleophiles (e.g., amines) on proteins.
m-Hydroxyphenyl azide (m-HPA) UV (≈350–365 nm) Hydroxy-substituted aryl-azide tuned for longer-wavelength activation.
Tetrafluorophenyl azide (TFPA) UV (≈350–365 nm) Electron-poor aryl azide; high nitrene yield; excellent for polymer/surface immobilization.
o-Nitrophenyl azide (o-NPA) UV (≈300–365 nm) Nitro-substituted aryl azide; useful where tuned reactivity and patterning are desired.
m-Nitrophenyl azide (m-NPA) UV (≈300–365 nm) Similar to o-NPA; choice depends on wavelength, matrix, and desired insertion profile.
Azidomethyl-coumarin UV (≈365–405 nm) Coumarin fluorophore with azide; allows photocrosslinking with built-in fluorescence.
L-photo-Leucine (diazirine amino acid) UV (≈350–365 nm) Metabolically incorporated diazirine AA; maps protein–protein contacts in live cells.
Technical Notes
  • Protect samples from light prior to activation; validate wavelength and exposure time.
Sample Submission
  • Indicate photo-crosslinker and target matrix (cell, membrane, surface).
Recommended Reading
  1. Wood CL, O’Dorisio MS. Covalent crosslinking of vasoactive intestinal polypeptide to its receptors on intact human lymphoblasts. J Biol Chem. 1985;260:1243–1247.
  2. Gomes AF, Gozzo FC. Chemical cross-linking with a diazirine photoactivatable cross-linker investigated by MALDI- and ESI-MS/MS. J Mass Spectrom. 2010;45:892–899.
  3. Krieg UC, Walter P, Johnson AE. Photocrosslinking of the signal sequence of nascent preprolactin to the 54-kilodalton polypeptide of the signal recognition particle. Proc Natl Acad Sci USA. 1986;83:8604–8606.

carbonyl-reactive cross linking, alkoxyamine aldehyde reaction, hydrazide reaction
Reagent Handles Notes / Applications
Aminooxy / Hydrazide –ONH₂ / –NHNH₂ Couple to oxidized sugars (–CHO) for uniform Fc labeling and polysaccharides.
Oxime / Hydrazone –C=N–O– / –C=N–NH– Often stabilized by reduction; gentle, aqueous conditions.
Technical Notes
  • Perform NaIO₄ oxidation on ice, in the dark; quench and polish rapidly.
Sample Submission
  • Provide glycoprotein ≥0.5–1.0 mg; specify desired Fc-label density.
Recommended Reading
  1. Byeon, J.Y., et al. (2010). Efficient bioconjugation of protein capture agents to biosensor surfaces using aniline-catalyzed hydrazone ligation. Langmuir 26(19):15430–15435.
  2. Dirksen, A., et al. (2006). Nucleophilic catalysis of hydrazone formation and transimination: implications for dynamic covalent chemistry. J. Am. Chem. Soc. 128(49):15602–15603.
  3. Dirksen, A.; Dawson, P.E. (2008). Rapid oxime and hydrazone ligations with aromatic aldehydes for biomolecular labeling. Bioconjugate Chem. 19(12):2543–2548.
  4. Bayer, E.A., et al. (1988). [Title not specified]. Anal. Biochem. 170:271–281.

Spacers are chains between reactive groups. Å-length sets reach, flexibility, and solubility. PEG linkers are short, defined PEGₙ segments with reactive ends for precise, hydrophilic coupling. PEG copolymers pair PEG with a second block to add stealth and materials functions such as self-assembly or hydrogel formation. Use a spacer for minimal mass and steric relief, a PEG linker for exact spacing and low fouling, and a PEG copolymer when you need carrier or delivery properties. 

PEG linker (generic): R1–O–(CH2–CH2–O)n–R2
Examples:
• NHS–PEGn–maleimide (amine ⇄ thiol)
• NHS–PEGn–DBCO (amine ⇄ SPAAC handle)
• NHS–PEGn–N3 (amine ⇄ azide handle)
• NH2–PEGn–COOH (amine ⇄ carboxyl)
• Biotin–PEGn–NHS (biotin tag ⇄ amine)

PEG copolymer (examples):
• PEG‑b‑PLA: R–O–(CH2–CH2–O)n–[O–CH(CH3)–C(=O)]m–R′
• PEG‑b‑PCL: R–O–(CH2–CH2–O)n–[O–(CH2)5–C(=O)]m–R′
• 4‑arm PEG (star): (X–O–(CH2–CH2–O)n–)4–Core
• PEG‑g‑(polymer): Backbone–{–g–O–(CH2–CH2–O)n–R}k

Notes: n, m, k = repeat counts. R/R′/X = reactive groups (NHS, maleimide, azide/alkyne, amine, COOH, etc.).
Spacer Type Typical Length / MW Flexibility Common Reactive Formats Use / Notes
Short alkyl (C2–C6) ~3–8 Å Rigid NHS–(alkyl)–maleimide; NHS–iodoacetyl Minimal distance; compact labeling on proteins/surfaces.
Medium/long alkyl (C8–C12) ~10–18 Å Rigid / hydrophobic Heterobifunctionals as above Greater reach; check aggregation risk in aqueous buffers.
Aromatic / phenylene Short (rigid) Rigid, planar e.g., aryl cores (SMCC-type) Orientation control; photo-stable linkers.
Peptide (Gly/Ser-rich) 5–20 aa (~19–76 Å extended) Flexible Terminal amine/acid; azide/alkyne Biocompatible, sequence-defined, protease-tunable.
Peptide (Pro/helix-forming) 5–20 aa More rigid As above Maintains separation without collapsing onto the target.
PEG linkers (PEG4–PEG24+) ~15–100 Å Flexible NHS, maleimide, azide/alkyne, DBCO Increase solubility; reduce non-specific interactions.
Discrete PEG (monodisperse) Exact PEGn length Flexible Heterobifunctional PEGn Precise spacing; clean LC/MS; batch-to-batch consistency.
Polymer PEG (linear) ~2–40 kDa Highly flexible coil mPEG-NHS, PEG-NHS, PEG-maleimide PEGylation; increases hydrodynamic size/“stealth”.
Branched / multi-arm PEG 2- or 4-arm (per-arm MW) Flexible 2-arm/4-arm PEG-NHS, -maleimide, -azide Multipayloads, scaffolds, hydrogel/crosslinking networks.
Zwitterionic spacers Variable Flexible Sulfobetaine / carboxybetaine with NHS/maleimide Very low fouling; serum-compatible interfaces.
Polysaccharide spacers (dextran) ~3–40 kDa Flexible Aminooxy/hydrazide; thiol-modified dextran Multivalent labeling; strong hydration layer.
POx / pHPMA (PEG alternatives) ~5–40 kDa Flexible Activated esters, maleimide, azide Hydrophilic “stealth” polymers when PEG isn't preferred.
Self-immolative (PAB) add-on N/A N/A VC-PAB / PABC modules Pair with spacers to enable triggered payload release (see Cleavable section).
Technical Notes
  • Select spacer length to balance solubility with target accessibility; avoid over-PEGylation that masks epitopes.
  • Discrete PEGn gives exact spacing; polymer PEG is specified by average MW (polydisperse).
Recommended Reading
  1. Hermanson, G.T. (2013). Bioconjugate Techniques, 3rd ed. Academic Press.
  2. Harris, J.M.; Zalipsky, S. (eds.) (1997). Poly(ethylene glycol): Chemistry and Biological Applications. ACS Symposium Series 680.
  3. Harris, J.M.; Kozlowski, A. (2001). Improvements in protein PEGylation: pegylated interferons for treatment of hepatitis C. J. Control. Release 72:217–224.
  4. Bentzen, E.L., et al. (2005). Surface modification to reduce non-specific binding of quantum dots in live cell assays. Bioconjugate Chem. 16:1488–1494.
  5. Lin, P.-C., et al. (2006). Ethylene glycol-protected magnetic nanoparticles for a multiplexed immunoassay in human plasma. Small 2(4):485–489.
  6. Zheng, M., et al. (2003). Ethylene glycol monolayer-protected nanoparticles for eliminating nonspecific binding with biological molecules. J. Am. Chem. Soc. 125:7790–7791.
  7. Verma, A.; Rotello, V.M. (2005). Surface recognition of biomacromolecules using nanoparticle receptors. Chem. Commun. 3:303–312.
  8. Kidambi, S., et al. (2004). Selective depositions on polyelectrolyte multilayers: self-assembled monolayers of m-dPEG acid as molecular template. J. Am. Chem. Soc. 126:4697–4703.

✂︎ 🔒 Type Common motifs / examples Trigger / mechanism Typical use
✂︎ Protease-cleavable Val–Cit–PABC; Val–Ala–PABC; PLGLAG (MMP); HSSKLQ (PSA) Cathepsin/MMP/PSA cleavage → self‑immolation ADC payload release
✂︎ Disulfide SPDP/LC‑SPDP; SPDB; PDP‑PEGn; hindered disulfide Reduction (GSH, intracellular) Redox‑triggered release
✂︎ Diselenide –Se–Se– Highly redox‑sensitive Fast redox response
✂︎ Hydrazone –C=N–NH– Acid‑labile (endosome/lysosome) pH‑triggered release
✂︎ cis‑Aconityl cis‑Aconityl amide Acid‑labile Prodrugs/ADCs
✂︎ Acetal/Ketal Benzylidene acetal; cyclic ketal Acid‑labile Endosomal release
✂︎ β‑Glucuronide / β‑Gal Ar‑O‑(glucuronide/galactoside) Glycosidase cleavage Tissue‑selective release
✂︎ Ester/Carbonate –C(=O)–O–; –O–C(=O)–O– Esterase hydrolysis Pro‑moieties
✂︎ Azo –N=N– Azoreductase/hypoxia Colon/hypoxia targeting
✂︎ ROS‑responsive Boronic ester; thioketal H₂O₂/ROS cleavage Inflamed/tumor sites
✂︎ Photolabile o‑Nitrobenzyl; nitroveratryl; coumarin Light‑triggered scission Spatiotemporal control
✂︎ Self‑immolative PAB/PABC modules Uncaps after primary trigger Clean payload release
🔒︎ Thioether Maleimide↔Cys (SMCC/MC); haloacetamide↔Cys Stable C–S bond Permanent labeling/ADCs
🔒︎ Click adducts 1,2,3‑Triazole (CuAAC/SPAAC); dihydropyridazine (IEDDA) Irreversible adducts Bioorthogonal, stable
🔒︎ Amide / Urea / Ether –C(=O)–NH– ; –NH–C(=O)–NH– ; –O– Chemically robust Imaging & standards
🔒︎ Oxime (neutral pH) –C=N–O– Generally stable Non‑releasing labels
🔒︎ Non‑cleavable PEG Linear/branched PEG without trigger No programmed cleavage Hydrophilic spacing
🔒︎ Rigid aryl/alkyl Phenylene, long alkyl spacers Inert scaffold Orientation/distance control
Technical Notes
  • Match release trigger (enzyme/redox/pH) to route and target compartment.
Sample Submission
  • Provide payload identity, desired release profile, and acceptable stability window.
Recommended Reading
Cleavable linkers
  1. Bargh, J.D.; Walsh, S.J.; Isidro‑Llobet, A.; Spring, D.R. (2019). Cleavable linkers in antibody–drug conjugates. Chemical Society Reviews. Link
  2. Balamkundu, S.; et al. (2023). Lysosomal‑cleavable peptide linkers in ADCs. Review. PMCID: PMC10669454
  3. Jeffrey, S.C.; et al. (2010). Expanded utility of the β‑glucuronide linker. ACS Med. Chem. Lett. PMCID: PMC4007898
  4. Lu, J.; et al. (2016). Linkers having a crucial role in ADCs. Int. J. Mol. Sci. Link
  5. Su, Z.; et al. (2021). Antibody–drug conjugates: recent advances in linker chemistry. Acta Pharm. Sin. B. PubMed
  6. Watanabe, T.; et al. (2024). Exo‑cleavable linkers: enhanced stability/therapeutic window. J. Med. Chem. DOI
Non‑cleavable linkers (exemplars & overviews)
  1. Lambert, J.M.; Chari, R.V.J. (2014). Ado‑trastuzumab emtansine (T‑DM1): an ADC with a non‑cleavable thioether linker. J. Med. Chem. DOI
  2. Donaghy, H. (2016). Effects of antibody, drug and linker on toxicities (incl. non‑cleavable). mAbs. PubMed
  3. Diamantis, N.; Banerji, U. (2016). ADCs—cleavable vs non‑cleavable linkers overview. Br. J. Cancer. Link
  4. Chari, R.; et al. / MCR Review (2020). Mechanisms for non‑cleavable vs cleavable linkers. Molecular Cancer Research. Link
  5. Tsuchikama, K.; An, Z. (2018). Conjugation & linker chemistries for effective ADCs. Protein & Cell. Link

Category Product (examples) Typical Formats Notes
Sortase A (LPXTG) Sortase A enzyme; LPETG-PEGn–Biotin/Dye; GGG-PEGn–NHS/–maleimide/–azide/–DBCO/–iodoacetamide PEG2/4/8/12/24; linear/branched LPXTG tag on protein + GGG-payload → site-specific fusion
Transglutaminase (TGase) mTG enzyme; Q-tag peptides; Cadaverine-PEGn–Biotin/Dye; PEG-diamines PEG4–PEG24; dyes/biotin/click handles Gln–Lys isopeptide formation under mild conditions
FGE / Aldehyde-Tag Aminooxy-PEGn–Biotin/Dye; Hydrazide-PEGn–Biotin/Dye; Aminooxy-PEGn–DBCO/BCN PEG4/12/24; heterobifunctional Oxime/hydrazone to engineered aldehyde (CXPXR)
BirA / AviTag BirA ligase (kit); AviTag control peptide; Desthiobiotin options Buffers, cofactors, QC controls Site-specific biotinylation on Lys within AviTag
Glycan-Directed Aminooxy-PEGn–Biotin/Dye; Hydrazide-PEGn–Biotin/Dye PEG4–PEG24; optional reduction step Periodate-oxidized glycans (Fc/PS)
Biotin/Streptavidin Targeting Biotin-PEGn–NHS / –maleimide / –azide / –DBCO PEG2–PEG24; linear/branched High-affinity capture or bridging
His-Tag / Ni-NTA NTA-PEGn–NHS / –maleimide PEG4/8/12 Reversible metal-chelate capture of His-tag proteins
Receptor-Directed Folate-PEGn–NHS/–azide; RGD-PEGn–NHS/–maleimide; GalNAc (mono/tri) linkers; Mannose-PEGn–NHS PEG4–PEG24; dye/drug/click variants Ligand-guided delivery (FR, integrins, ASGPR, MR)
PSMA Targeting PSMA (Glu–urea–Lys)–PEG4/8/12–NHS; PSMA–PEGn–azide; PSMA–PEGn–maleimide PEG4–PEG24; linear/branched Ligand-directed delivery to PSMA-positive cells
Cysteine-Directed (Re-bridging) Dibromomaleimide–PEGn–NHS/–azide; Bis-maleimide–PEGn–(X); PD-PEGn–maleimide PEG2/4/8/12/24; heterobifunctional Site-selective disulfide re-bridging for homogeneous placement
PTAD (Tyr-Selective) PTAD–PEG4/8–NHS; PTAD–PEGn–azide; PTAD–PEGn–DBCO PEG4–PEG24; linear Tyrosine-specific labeling via ene reaction
PROTAC Couplers VHL-ligand–PEG3/4–NHS/–azide/–alkyne; CRBN-ligand–PEGn–NHS/–azide; NHS–PEGn–alkyne; Azido–PEGn–amine PEG2–PEG24; mono- or heterobifunctional Handles and spacers for assembling heterobifunctional degraders
Technical Notes
  • Use engineered tags for precise placement; validate activity post-conjugation.
Sample Submission
  • Submit sequence, tag type (LPXTG/Q-tag/FGE/Avi), and target ligation plan.
Recommended Reading
  1. Agarwal, P. & Bertozzi, C.R. (2015). Site-specific antibody–drug conjugates: the nexus of chemistry and biology. ACS Cent. Sci. 1:287–297.
  2. Boutureira, O. & Bernardes, G.J.L. (2015). Advances in chemical protein modification. Chem. Rev. 115:2174–2195.
  3. Popp, M.W. & Ploegh, H.L. (2011). Making and breaking peptide bonds: protein engineering using sortase. Angew. Chem. Int. Ed. 50:5024–5032.
  4. Guimaraes, C.P. et al. (2013). Site-specific C-terminal labeling of proteins using sortase. Nat. Protoc. 8:1787–1799.
  5. Theile, C.S. et al. (2013). Site-specific N-terminal labeling of proteins using sortase-mediated reactions. Nat. Protoc. 8:1800–1807.
  6. Jeger, S. et al. (2010). Site-specific and stoichiometric modification of antibodies by bacterial transglutaminase. ChemBioChem 11:1738–1746.
  7. Carrico, I.S. et al. (2007). Introducing genetically encoded aldehydes into proteins. Nat. Chem. Biol. 3:321–322.
  8. Wu, P. et al. (2009). Site-specific chemical modification of recombinant proteins in mammalian cells using the aldehyde tag. Proc. Natl. Acad. Sci. USA 106:3000–3005.
  9. Beckett, D. et al. (1999). A minimal peptide substrate for BirA-catalyzed biotinylation. Protein Sci. 8:921–929.
  10. Howarth, M. & Ting, A.Y. (2008). Site-specific labeling of proteins with biotin: biotin ligase-tag technology. Nat. Protoc. 3:534–545.
  11. Zuberbühler, K. et al. (2014). Antibody glycoengineering for site-specific conjugation. mAbs 6:844–852.
  12. Junutula, J.R. et al. (2008). Site-specific conjugation of a cytotoxic drug to a THIOMAB antibody. Nat. Biotechnol. 26:925–932.
  13. Axup, J.Y. et al. (2012). Synthesis of site-specific antibody–drug conjugates using unnatural amino acids. Proc. Natl. Acad. Sci. USA 109:16101–16106.

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FAQ

Why does linker selection matter in ADCs?

Linker choice governs plasma stability, pharmacokinetics, therapeutic index, and the mechanism/timing of payload activation—so it directly shapes overall ADC performance.

How does linker choice affect plasma stability?

A stable linker prevents premature drug release in blood, maintaining ADC integrity until it reaches target cells.

What is the impact on pharmacokinetics?

Hydrophilicity, charge, and sterics from the linker modulate circulation half‑life and clearance, influencing exposure at the target site.

How does linker selection influence therapeutic index?

By controlling when and where the payload is released, the linker helps maximize tumor killing while minimizing effects in healthy tissues.

Cleavable vs non‑cleavable—what’s the difference?

Cleavable linkers release the payload in response to triggers (e.g., enzymes, pH, redox), while non‑cleavable linkers retain a stable attachment and rely on intracellular degradation; this choice defines the ADC’s core release strategy.

What role does a PEG spacer play?

PEG segments increase aqueous solubility, reduce non‑specific interactions/aggregation, and can lower immunogenicity risk, improving manufacturability and in vivo behavior without changing the fundamental release mechanism.

What’s the difference between a spacer, a PEG linker, and a PEG copolymer?

Spacers are short chains that set reach and orientation; PEG linkers are defined PEGn segments with reactive ends for hydrophilic, precise spacing; PEG copolymers combine PEG with a second block (or multi‑arm core) to add stealth, self‑assembly, or hydrogel behavior.

How long is PEG4, PEG12, or PEG24?

Approximate end‑to‑end spacing: PEG4 ≈ ~15 Å, PEG12 ≈ ~45 Å, PEG24 ≈ ~90–100 Å (buffer and conformation dependent).

When should I choose a cleavable linker?

Use cleavable linkers when payload release at the target is required—e.g., protease motifs (Val‑Cit/Val‑Ala + PABC), disulfide for redox, hydrazone/cis‑aconityl for acid, glucuronide/galactoside for enzymes, or photolabile/ROS‑responsive systems.

What determines linker selection?

Reactive groups (amine/thiol/azide/aldehyde), sterics, solubility, release needs, and downstream use (in vitro vs in vivo).

Do you provide method transfer?

Yes—SOPs, batch records, CoAs, and data packages for tech transfer and filings.

Can you handle nanobody/site-specific designs?

Yes—Cys engineering, Sortase/TGase tags, and glycan strategies with optimized linkers.

When is a non‑cleavable linker preferred?

Choose non‑cleavable for permanent labeling or stable constructs—e.g., thioether (maleimide↔Cys), click adducts (triazole/IEDDA), robust amide/urea/ether, or neutral‑pH oxime.

Which chemistries are best for thiol (Cys) coupling?

Common options: maleimide (fast, near‑neutral pH), haloacetyl (iodo/bromo/chloro‑acetamide; irreversible S‑alkylation), pyridyl disulfide (reversible S–S), vinyl sulfone (stable thioether via Michael addition), and epoxide (broad reactivity at higher pH).

Are PEG‑Br/PEG‑Cl/PEG‑I or PEG‑tosylate thiol‑reactive?

They’re building blocks (leaving‑group PEGs) used to synthesize functionalized PEGs via SN2—useful in organic media but not selective bioconjugation handles by themselves.

What buffer and pH should I use for NHS‑ester reactions?

Use PBS/HEPES around pH 7.2–7.5; avoid Tris or glycine (they consume NHS esters). LC/PEG spacers can reduce steric clash on crowded surfaces.

What conditions are best for maleimide–thiol coupling?

Couple near pH 6.5–7.5, include 1–2 mM EDTA, and exclude reducing agents; at higher pH, maleimides can add to amines. Optional ring‑opening post‑coupling can enhance long‑term stability.

How do I label glycans/aldehydes on proteins?

Oxidize glycans to aldehydes (NaIO4 on ice, in the dark), then couple with aminooxy or hydrazide linkers to form oxime/hydrazone (often stabilized by reduction).

What’s the difference between discrete (monodisperse) PEG and polymer PEG?

Discrete PEGn gives exact, LC/MS‑clean spacing; polymer PEG is specified by average MW (2–40 kDa) and used for PEGylation to increase hydrodynamic size and reduce fouling.

Are there PEG alternatives for “stealth” spacing?

Yes—poly(2‑oxazoline) (POx) and pHPMA are hydrophilic alternatives available with similar activated ends (esters, maleimide, azide).

Which click strategy should I pick?

CuAAC (azide+alkyne+Cu) is quantitative but not for live systems; SPAAC (DBCO/BCN+azide) is copper‑free for live/in vivo; IEDDA (TCO+tetrazine) is ultra‑fast and orthogonal for dual systems.

Do you support site‑specific conjugation systems?

Yes—Sortase A (LPXTG↔GGG), microbial transglutaminase (Gln–Lys), FGE/Aldehyde‑Tag (oxo‑handles for oxime/hydrazone), and BirA/AviTag (enzymatic biotin) with PEG‑functional payloads.

How do I choose spacer length?

Balance reach and solubility with epitope access; use short alkyl/aryl for rigid, minimal distance; PEGn or peptide for flexible, hydrophilic spacing; avoid over‑PEGylation that masks binding.

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