HO-PEG5K-NHS - average Mₙ 5,000

Cat. No.: H478170

GRADE & PURITY average Mₙ 5,000

Synonyms

Hydroxyl-PEG-NHS Ester, HO-PEG-NHS

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

Overview

Description

Hydroxyl-PEG-NHS Ester (HO-PEG-NHS) is hydrophilic in nature.HO-PEG-NHS may be conjugated on to a hydrophobic H40-PLA-COOH molecule to form H40-PLA-PEG-Apt) (aptamer-conjugated). Other applications may include: bioconjugation, drug delivery, PEG hydrogel, crosslinker, and surface functionalization.Applications may include: bioconjugation, drug delivery, PEG hydrogel, crosslinker, and surface functionalization

Specifications

Synonyms

Hydroxyl-PEG-NHS Ester, HO-PEG-NHS

Specifications & Purity

average Mₙ 5,000

Legal Information

Product of JenKem Technology

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

This product requires cold chain shipping. Ground and other economy services are not available.

Application Protocols

Example protocol (general, research use only): Lysine-selective PEGylation of a protein with HO-PEG5K-NHS

• Materials:

  • Protein of interest in phosphate buffer (50 mM, pH 7.8–8.2, 100–150 mM NaCl), free of primary amine buffers.
  • HO-PEG5K-NHS.
  • Anhydrous DMSO or DMF.
  • Quench: ethanolamine or Tris base.
  • Desalting column or ultrafiltration device (e.g., 30 kDa MWCO).

• Steps:

  1. Equilibrate all reagents to room temperature in a desiccator to avoid condensation. Prepare a fresh 50 mg/mL stock of HO-PEG5K-NHS in anhydrous DMSO.
  2. Adjust the protein solution to pH 7.8–8.2. Typical protein concentration: 1–5 mg/mL.
  3. Add the PEG stock dropwise to the protein solution while gently mixing to reach 5–10 molar equivalents of PEG per protein. Keep final DMSO ≤10% v/v.
  4. Incubate 1 h at room temperature. Monitor progress by sampling and assessing free amines (e.g., TNBS assay) or by analytical SEC.
  5. Quench with 20 mM ethanolamine (final) for 10–15 min.
  6. Remove excess reagents by SEC or ultrafiltration (3–5 diafiltration volumes).
  7. Analyze the product by SDS-PAGE (mobility shift), SEC (elution volume shift), and UV/Vis for recovery.

• Notes:

  • Avoid buffers containing Tris/glycine during coupling.
  • If solubility issues arise, raise ionic strength or add 2–5% DMSO.
  • Optimize stoichiometry/time to tune degree of labeling.

Surface coupling sketch (general): Amine-functionalized substrate in dry DMF with a hindered base (e.g., DIPEA), add HO-PEG5K-NHS solution, react 1–2 h at RT, rinse thoroughly, and post-functionalize the residual hydroxyl as desired.

Biological Roles

Context (general knowledge; no therapeutic claims):

• PEG is considered biologically inert and highly hydrophilic. When grafted to biomolecules or surfaces, PEG chains create a hydration shell that reduces protein adsorption and can diminish nonspecific interactions in vitro.
• As a spacer, a ~5 kDa PEG introduces substantial hydrodynamic radius and conformational flexibility, often improving solubility and reducing aggregation of conjugates in biochemical assays.
• NHS-terminated PEG reagents are tools for covalent modification of primary amine-containing biomolecules (e.g., lysine side chains) in research settings, enabling attachment of PEG to modulate biophysical properties.
• The terminal hydroxyl group provides a handle for further functionalization (e.g., conversion to azide/alkyne for bioorthogonal labeling), allowing construction of complex, multifunctional biomolecular assemblies.

Limitations and considerations:

• Labeling via NHS is not site-specific for proteins; it targets accessible, sufficiently nucleophilic primary amines. Resulting products are typically heterogeneous in label position and degree.
• Over-PEGylation can impair biochemical activity. Empirical optimization of pH, stoichiometry, and time is necessary to balance modification extent and function.
• Hydrolysis of active esters competes with conjugation in aqueous media; rapid mixing and controlled pH are essential to maintain efficiency.

Note: All roles described are for research and laboratory investigations only; not for clinical or diagnostic use.

Buffer Applications

This product is a heterobifunctional PEG linker and not a buffering agent. It does not constitute a defined acid/base pair with a target pH range.

Practical guidance related to buffers during use (general):

• Use amine-free buffers (e.g., phosphate, HEPES, bicarbonate) for NHS-mediated coupling. Avoid Tris, glycine, and other primary amine-containing buffers until quenching.
• Typical working pH: 7.5–8.5 (literature) to balance amine nucleophilicity and NHS stability.
• Ionic strength: moderate salt (e.g., 100–200 mM NaCl) can help maintain protein solubility; verify compatibility for your system.

Recipes are application-specific and should be optimized experimentally for the target biomolecule or surface.

Green Alternatives

Sustainability considerations for handling NHS–PEG reagents (general guidance):

Greener solvent choices and practices:

• Prefer anhydrous ethanol or isopropanol as rinse/cleaning solvents when compatible, reducing use of higher-toxicity aprotics.
• For stock preparation, DMSO is generally considered to have a more favorable environmental and health profile than DMF or NMP; validate compatibility with your system.
• Minimize solvent volumes by preparing concentrated stocks and immediate use, thereby reducing waste.

Comparison (qualitative; literature-based):

• DMSO vs. DMF:
  • EHS profile: DMSO typically greener than DMF (reprotox concerns with DMF).
  • Reactivity: both suitable; DMSO is more hygroscopic—use freshly dried material.
• Acetonitrile vs. NMP:
  • EHS: acetonitrile has lower chronic toxicity but is flammable; NMP has reproductive toxicity flags.
  • Solubility: PEG5K often dissolves better in NMP/DMF than in MeCN; balance EHS with solubility needs.

Process intensification:

• Conduct couplings at room temperature to avoid energy-intensive heating and to limit NHS decomposition.
• Use aqueous buffers with minimal organic co-solvent where biomolecule stability permits, accepting somewhat faster hydrolysis in exchange for greener media.

Waste reduction:

• Quench residual active ester with benign nucleophiles (e.g., ethanolamine) before disposal.
• Employ size-exclusion chromatography or ultrafiltration with water-based eluents to avoid large volumes of organic waste.

Pharmaceutical Uses

No clinical or therapeutic claims are made. The following information concerns formulation/manufacturing contexts in research and development (general knowledge):

• PEGs are widely used as excipients (e.g., solvents, plasticizers) and as polymeric backbones for conjugates. However, NHS-activated PEGs are reactive intermediates intended for conjugation, not as final excipients.
• For process development, heterobifunctional PEGs like HO-PEG5K-NHS may serve as linkers/spacers in the synthesis of PEGylated research materials, affinity media, and prototype delivery systems.
• Regulatory status: Only non-reactive PEGs of defined grades are covered by pharmacopoeias. Activated PEGs (e.g., NHS esters) typically lack compendial monographs and are handled as process reagents. Any cGMP or clinical manufacturing use would require supplier qualification, impurity profiling, and stability data specific to the lot.
• Residuals and extractables: control of free NHS, succinimide, solvents, and activation byproducts is essential for sensitive applications; review CoA and perform in-house QC as needed.

Conclusion: In pharmaceutical R&D settings, HO-PEG5K-NHS would primarily be used upstream as a conjugation intermediate under research-use restrictions.

Physical Properties

Item-specific properties (from Product Data):

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.

General/literature properties for PEG5K NHS-type reagents (for planning purposes only; not item specifications):

• Average molecular weight: ~5,000 g/mol (nominal; actual Mn/Mw and Ð depend on lot and manufacturer).
• Physical form: typically white to off-white solid; can be amorphous or waxy depending on chain length and end-groups.
• Solubility: high in polar aprotic solvents (DMF, DMSO, acetonitrile) and many alcohols; variable in water due to NHS hydrolysis risk—brief aqueous exposure at neutral to mildly basic pH is common during coupling.
• Hygroscopicity: PEG chains are hygroscopic; NHS esters are moisture-sensitive and hydrolyze to the corresponding acid (literature).
• Thermal behavior: softening rather than a sharp melt; glass transition for PEGs occurs below room temperature for low MW but rises with MW. NHS functionality decomposes/hydrolyzes upon prolonged heating or in moisture (literature).

Optical/refractive, pKa/logP, and precise thermal constants:

• Not typically defined for polydisperse PEG reagents with reactive termini; values vary and are not generally useful as specifications.

Practical implications:

• Prepare stocks in anhydrous DMF/DMSO and use promptly.
• Minimize exposure to aqueous buffers until immediately before conjugation to limit NHS hydrolysis.

Quality & Grades

Item-specific quality details:

• Grade/Purity: Not specified for this item; refer to CoA/Spec Sheet.

How to interpret quality for heterobifunctional PEG linkers (general guidance):

• Functionalization efficiency: the fraction of chains bearing the intended end-groups (NHS on one end, –OH on the other). High end-group fidelity is critical for predictable conjugation.
• Dispersity (Ð = Mw/Mn): PEGs are polydisperse; narrower Ð yields more uniform conjugates and predictable hydrodynamic size. Lot CoA often reports Mn (or nominal MW) and sometimes Ð.
• Residuals: low levels of free NHS, succinimide, starting alcohols/acids, and metal catalysts are preferred for sensitive bioconjugations. Check CoA for residual solvent and impurity limits when available.
• Hydrolysis profile: NHS content can decline with improper storage. Recently manufactured and properly stored lots show stronger coupling performance.
• UV/LC suitability: Some vendors denote bioconjugation or crosslinking grade, implying lower UV-absorbing impurities and higher end-group integrity. Without an explicit grade, verify compatibility in your assay.

Recommendations:

• Request/consult the lot-specific CoA for Mn, functional group assay, and residuals.
• For quantitative applications (e.g., degree of labeling), perform an active ester content test or a small-scale coupling control to confirm reactivity.
• If working under regulated environments, confirm whether the material meets internal specs for bioconjugation grade or higher purity requirements.

Reaction & Applications

Applicable chemistries (general/literature for NHS–PEG linkers):

• Amine coupling: NHS esters react with primary aliphatic amines to form stable amide (or carbamate if carbonate-linked) bonds. Typical substrates: lysine residues on proteins/peptides, amine-functionalized surfaces/particles, and small-molecule amines.
• Surface modification: imparts hydration, antifouling, and spacing effects on nanoparticles, resins, and planar substrates while leaving an –OH for secondary derivatization.
• Spacer/solubilizer: the PEG5K chain increases aqueous solubility and reduces aggregation in conjugates.

Use cases in materials and chemical biology (no clinical claims):

• Protein PEGylation (lysine-selective) to modulate hydrodynamic size and reduce nonspecific interactions in analytical workflows.
• Creation of amphiphilic constructs by post-functionalizing the hydroxyl terminus (e.g., to azide/alkyne for click chemistry, tosylate for nucleophilic substitution, or CDI-activated carbonate for further amine coupling).
• Crosslinking workflows when combined with a second reactive partner on the –OH end, enabling heterobifunctional bridges between amines and other nucleophiles.

Practical notes:

• NHS half-life decreases with increasing pH and temperature; perform couplings at pH 7.5–8.5 and ambient temperatures to balance rate and hydrolysis.
• Use molar excesses of linker relative to available amines (commonly 2–20 equiv per target amine for surfaces; protein labeling often 2–10 equiv per lysine or 2–20 equiv per protein depending on desired DOL).
• Quench unreacted NHS with ethanolamine or Tris after coupling (avoid amine-containing buffers during the coupling step).
• Confirm conjugation via SEC, SDS-PAGE shifts, MALDI/ESI-MS (if mass resolution allows for polymer dispersity), or colorimetric assays for residual active ester (e.g., TNBS on free amines).

Reaction Conditions

Typical conditions for NHS–amine coupling with PEG linkers (general literature guidance; adjust per system):

• Solvent system: aqueous buffer (phosphate, HEPES, bicarbonate) at pH 7.5–8.5 with 0–10% v/v anhydrous DMSO or DMF cosolvent. Avoid primary amines in the buffer during coupling.
• Temperature/time: 20–25°C for 0.5–2 h for many proteins/surfaces. More hindered amines may require longer times or slightly elevated temperature (≤30°C) to minimize hydrolysis.
• Stoichiometry: 2–20 equivalents of HO-PEG5K-NHS per target amine; for proteins, 2–20 equivalents per protein are commonly screened to tune degree of labeling.
• Concentrations: protein 0.5–10 mg/mL; PEG linker 1–50 mg/mL stock in anhydrous DMSO/DMF, added to achieve desired molarity.
• Catalysis/base: additional base often unnecessary above pH 8.0. For small-molecule amines in organic media, tertiary amines (e.g., DIPEA) can be used cautiously to scavenge acid without promoting hydrolysis excessively.
• Quench: ethanolamine or Tris (10–100 mM) after the desired reaction time.

Kinetic notes (literature):

• NHS ester half-life at 25°C is on the order of tens of minutes to a few hours in aqueous buffers between pH 7.5–8.5; higher pH/temperature accelerates hydrolysis relative to amine coupling.

Workup and analysis:

• Remove excess PEG and byproducts via SEC, dialysis/UF (e.g., 10–30 kDa MWCO), or precipitation. Analyze by SEC, SDS-PAGE shift, and if applicable, MS or NMR of small-molecule conjugates.

Caveats:

• The exact reactivity depends on whether the NHS is linked via ester vs. carbonate; both behave as acylating agents but differ slightly in stability. Verify with supplier documentation if needed.

Safety & Handling

Authoritative data:

• GHS classification, pictograms, signal word, and H-statements: Not specified for this item; refer to SDS.

General safety guidance for NHS-activated PEG reagents (literature/practice; defer to SDS):

• Hazards: NHS esters are irritants and moisture-sensitive; hydrolysis liberates NHS (irritant). PEG backbones are generally of low acute toxicity but can cause irritation as dust/particulate.
• PPE: wear lab coat, safety glasses, and appropriate chemically resistant gloves (e.g., nitrile). Handle powders in a fume hood to avoid inhalation of dust/aerosols.
• Handling: work under dry conditions. Use anhydrous solvents and desiccated glassware. Cap containers promptly; consider nitrogen/argon backfill for aliquots.
• Incompatibilities: water, strong bases (accelerate NHS hydrolysis), strong nucleophiles, and primary amines unless performing the intended coupling. Avoid prolonged exposure to elevated temperatures.
• First aid (overview; consult SDS):
  • Skin/eye contact: rinse with water for at least 15 minutes; remove contaminated clothing; seek medical advice if irritation persists.
  • Inhalation: move to fresh air; seek medical attention if symptoms occur.
  • Ingestion: rinse mouth; do not induce vomiting; seek medical attention.
• Spill response: avoid dust formation; collect mechanically; wipe residues with damp disposable towels or appropriate solvent while controlling exposure; dispose per local regulations.
• Waste: aqueous/organic waste streams containing NHS esters should be quenched (e.g., with ethanolamine or Tris) before disposal per institutional guidelines.

Solvent Selection

Polarity and miscibility (general/literature for PEG linkers):

• PEG5K derivatives dissolve well in polar aprotic solvents (DMF, DMSO, NMP, acetonitrile). Limited solubility in nonpolar solvents (hexanes, toluene) without co-solvent. Aqueous solubility can be high, but NHS hydrolyzes in water.

Recommended use patterns:

• Stock solutions: prepare fresh, anhydrous stocks in DMF or DMSO (10–100 mg/mL), filter (0.22 µm) if needed, and use immediately.
• Aqueous coupling: add the anhydrous stock to buffered amine solutions shortly before use. Maintain pH 7.5–8.5 to balance amine nucleophilicity vs. NHS hydrolysis (literature).
• Protein work: limit organic co-solvent to ≤10% v/v to maintain protein stability; screen DMSO vs. DMF tolerance.

Quick comparison (general considerations):

• DMF: excellent solvent power; low volatility; water-miscible; can contain amine impurities—use high-purity, amine-free grades.
• DMSO: broadly compatible with biomolecules; hygroscopic; can slow hydrolysis versus purely aqueous media by lowering water activity.
• Acetonitrile: good for some surface modifications; lower PEG solubility at high MW; volatile.
• Water/buffers: operationally convenient but rapidly hydrolyze NHS; use only during the coupling window at controlled pH.

Practical tips:

• Dry solvents over molecular sieves (3Å/4Å) and validate water content if your workflow is moisture-sensitive.
• Pre-wet reaction surfaces with the selected solvent to minimize localized high concentrations that can precipitate PEG.
• Avoid primary amines in buffers (e.g., Tris, glycine) during coupling; use phosphate, HEPES, or bicarbonate instead.

Storage & Reconstitution

Item-specific storage/shipping (from Product Data):

• Storage conditions: Store at -20°C.
• Shipped in: Ice chest + Ice pads.

General best practices for NHS–PEG reagents:

• Protect from moisture and light; store in tightly sealed, inert-atmosphere containers within a desiccator. Allow vials to warm to room temperature inside a desiccator before opening to prevent condensation.
• Aliquot on receipt into single-use portions to minimize freeze–thaw and headspace moisture ingress.

Reconstitution (general guidance):

• Solvents: anhydrous DMSO or DMF are recommended for stock solutions. Prepare immediately before use.
• Typical stock concentrations: 10–100 mg/mL depending on application and viscosity. Pass through a 0.22 µm filter if needed for bioconjugation workflows.
• Aqueous use: add the anhydrous stock to the aqueous reaction buffer immediately prior to coupling. Avoid storing aqueous solutions of NHS-PEG; hydrolysis will occur.

Short-term storage of solutions (if unavoidable):

• Anhydrous DMSO/DMF stocks may be stored at -20°C under inert gas for a short period (e.g., days) but activity decays with time and residual moisture. Validate activity before critical experiments.

Disposal:

• Quench any remaining active ester (e.g., with ethanolamine) prior to disposal following institutional and local regulations.

Note: For exact shelf life, residual solvent levels, and functional group assay, consult the lot-specific CoA/Spec Sheet.

Structure & Identity

Brief overview: HO-PEG5K-NHS is a heterobifunctional polyethylene glycol (PEG) reagent bearing a terminal hydroxyl (–OH) group on one end and an N-hydroxysuccinimide (NHS) activated ester on the other, enabling selective amine coupling while retaining a free hydroxyl for subsequent derivatization.

• SKU: H478170
• Product name: HO-PEG5K-NHS
• Category: Materials Science (research reagent)
• Research use: For research use only

Item-specific identifiers (from Product Data):

• CAS: Not specified for this item; refer to CoA/Spec Sheet.
• InChIKey: Not specified for this item; refer to CoA/Spec Sheet.
• SMILES: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular formula: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.

Structural features (general/literature description for PEG5K NHS reagents):

• Polyether backbone comprised of repeating –CH2–CH2–O– units (polydisperse). For an average 5 kDa PEG, the chain corresponds to roughly 110–115 ethylene oxide units (approximate, literature).
• One terminus: N-hydroxysuccinimide activated ester (typically a succinimidyl ester of a PEG terminal carboxylate or carbonate) that reacts with primary aliphatic amines to form stable amide (or carbamate) linkages (literature).
• Opposite terminus: free hydroxyl (–OH), enabling further activation (e.g., tosylation, CDI-carbonate formation) or conjugation strategies.
• 2D description in words: a flexible, linear poly(ethylene glycol) chain with an NHS-activated acyl functionality at one end and a terminal hydroxyl at the other; no defined stereocenters; no aromatic rings except the succinimide moiety within the NHS leaving group.

Notes on identifiers for polymers:

• PEG reagents are polydisperse; exact SMILES/InChIKey are typically undefined or represent an idealized repeat unit and are not generally assigned for catalog items. Refer to the CoA for lot-specific Mn and dispersity (Ð) if provided.

Synthetic Utility

Functional groups and reactivity (general/literature):

• NHS-activated terminus: acylating agent for primary aliphatic amines, forming amide (or carbamate if carbonate-linked) bonds under mild conditions.
• Hydroxyl terminus: can be transformed into a wide range of handles—tosylate/mesylate (for SN2), azide/alkyne (click chemistry), CDI-carbonate (amine coupling), aldehyde (via periodate after diol introduction), carboxyl (via TEMPO oxidation after appropriate modification), etc.

Synthetic value:

• Heterobifunctional spacer: enables sequential, chemoselective attachment of different entities to construct conjugates, dendrimers, surfaces, or block copolymers.
• Solubilizing handle: incorporation of PEG5K markedly increases water compatibility and can improve reaction homogeneity for hydrophobic partners.
• Protection/stealth: PEG segments can mask charge/hydrophobicity in materials science constructs, tuning interfacial properties.

Representative transformations:

• NHS-mediated coupling to amine-bearing ligands, followed by conversion of the free –OH to an azide and CuAAC to an alkyne-tagged partner.
• Activation of –OH with CDI to form an activated carbonate, enabling a second amine coupling step to generate di-functional architectures.
• Surface immobilization: first anchor the NHS end to an amine-functionalized substrate, then derivatize the distal –OH to introduce detection tags or reactive groups.

Practical considerations:

• Sequence planning: perform NHS-amine coupling before manipulations that require strong base or aqueous conditions to avoid premature hydrolysis.
• Purification: size-exclusion chromatography, ultrafiltration (MWCO ~10–30 kDa), or precipitation from non-solvents (e.g., cold ether) are common for PEG constructs.

Target Specificity

Not applicable. This product is a chemical linker reagent and not a biological targeting molecule or antibody. No antigen/epitope, species reactivity, clone, or isotype information applies.