purpose for the HF cleavage and protection from resins in organic chemistry

1. HF Cleavage And Deprotection From
Resins.
Guided by Prepared by
Dr. Hemke sir Miss. Shweta Raut
M.Pharm 2nd sem.(2019-2020)
Pharmaceutical Chemistry Department
SKB College Of Pharmacy, Kamptee.

2. What is HF cleavage?
In this process, ayhydrous hydrogen fluoride(HF) is used
to remove the side chain protecting groups of the
assembled peptide and to release the resin, is known as
HF cleavage.
• With the help of Hydrogen fluoride(HF) breaking the bond.

3. INTRODUCTION
On completion of chemical synthesis of the peptide chain, the
final step requires the removal from the solid-phase support
and liberation of the protected side chains of the trifunctional
amino acids (I). Many different approaches to this problem
have been established, but the procedure most widely used
for all Boc (butoxycarbonyl)/Bzl (benzoyl chloride)-based
peptides has been treatment with liquid HF.
ADVANTAGE:
HF is a strongly protonating acid(Ho=-10,8)
PROPERTY:
• Nonoxidizing
• Excellent solvent for peptide

4. one major drawback of using HF is the number of deleterious
side reactions that are promoted by the SN1 removal of
protecting groups. The solvolysis reaction generates benzyl
carbocations that are stabilized by HF. These carbocations are
potent alkylating species of amino acids with nucleophilic side
chains, such as cysteine, methionine, tryptophan, and tyrosine.
The addition of compounds, such as anisole, dimethyl sulfide,
and p-cresol, to the reaction mixture helps to minimize these
reactions by scavenging these carbocations. Several of the
most commonly used scavengers in Boc/Bzl-based strategies
are shown in Table 1.

6. MATERIALS
1. HF cylinders may be purchased from Matheson Gas Company
(Newark, NJ). This material is highly corrosive and toxic. HF dissolves
glass and must be handled in an HF-resistant apparatus in a scrubber
equipped fume hood.
2. A suitable HF-resistant cleavage apparatus may be purchased from
Immuno-dynamics.
3. It is recommended that all users keep the HF antidote calcium
gluconate in the laboratory where HF is handled.
4. A wide variety of scavengers are routinely used in HF reactions.
Anisole, p-cresol,p-thiocresol,dimethylsulfide,1,2ethanedithiol &
thiophenol.
5. The solvents used for extraction may be obtained from Fisher Scientific,
These solvents include diethyl ether, ethyl acetate, and glacial acetic acid.

7. METHODS
Successful cleavage and deprotection of the final peptide product rep-
resent one of the most crucial steps in the synthetic cycle. Poor cleavage
procedures greatly increase the work-up time required to isolate the
product because of the increased complexity of the crude product
mixture. In general, several protecting groups may be removed directly
from the resin-bound peptide prior to cleavage. Selection of the proper
scavenger cocktail is also crucial to minimize deleterious side reactions
during the HF cleavage. Finally, the time and temperature are two
variables that can be easily modified if incomplete deprotection of the
peptide appears to be a problem. present general protocols for standard
and low-high HF cleavage procedures.
Preclaving procedure
1. Removal of N-terminal Boc Group
2. Deformylation of Trp
3. Removal of Dnp from Histidine

8. Removal of N-terminal Boc Group
Removal of the N-terminal Boc group eliminates the deleterious t-butyl
carbocation from being generated in the HF reaction. This carbocation is
a potent alkylator of amino acids with nucleophilic side chains.
1. Add the peptide resin to either a round-bottom or Erlenmeyer flask
with a stir bar.
2. Add 50% (v/v) trifluoroacetic acid/dichloromethane to the flask
(approx 20 mL/g of resin). For peptides containing reduced Met or
unprotected Trp, dithiothreitol or methylindole may be added at a
concentration of l-5% wt/vol to the deblocking solution to prevent
oxidation of these side chains.
3. Allow reaction to evolve CO2 for several minutes before capping.
Place the flask on a magnetic stir plate and allow to mix for 30 min.

9. 4. Filter the resin through a fretted-glass funnel to remove the trifluoro-
acetic acid, and wash the resin several times with dichloromethane.
Some researchers also perform a base-wash following the deblocking to
remove the TFA salt using 10% TEA m DCM.
5. Wash the resin with methanol several times followed by
dichloromethane and then methanol last. Some laboratories prefer a
final DCM wash to leave the resin “fluffy” as a means of improving the
HF and anisole permeation of the resin.
6. Dry the resin overnight m a vacuum desicator.

10. Standard HF procedure
1. a portion of the final deblocked peptide resin in placed into a dry Kel-F
reaction vessel with dry Teflon TM ~ coated magnetic stir bar. Resins
quantity limited
2.anisole is pippeted into the reaction vessel at a ratio 1ml/g of resin it is
placed into a HF apparatus .
3.raction vessel is cooled with dry ice ethanol.
4. then closing the valve to the aspirator. Distillation time required
depends on the amount of HF needed.
5.the reaction vessels is now placed into an ice water bath & allow to
warm upto 0-4degree C.
6. Stirring of the resin-HF- anisole slurry must be through, at the end of
the reaction time removal of HF is by evaporation
7. Continue a trap in between the vacuum reaction vessels is cooled
with dry ice-ethanol thermos for 10-15 min.
8. Trap is cold lose the valve to the water aspirator ,close the valve
system & open valve to the vacuum pump slowly open to the system
9.residual HF system upto 90 min removed.

12. Strategies to minimize side reactions
1. As a standard rule, we only cleave a small portion of the
completed peptide resin, usually one-third or less, until suitable
cleavage conditions have been established.
2. For the standard peptide containing no difficult residues, the 1
part anisole or p- or m-cresol to 1 part resin to 9 parts HF works
quite well.
3. Lowering the temperature to between -10 and -5°C by
incorporating a salt-ice bath will slow down aspartimide
formation and anisylated glutamic acid.
4. For peptides containing Cys (Meb), we routinely use 1 part p-
cresol to 1 part p-thiocresol to 18 parts HF (v/v)/g of resin (21).
5. For peptides containing Met, addition of dimethyl sulfide helps
prevent alkylations of methionine by competing for
carbocations.

13. Low and high HF procedure
1. Place l-2 g of peptide resin and a stirring bar m the cleavage vessel.
Use the following ratio of scavengers per gram of resin: 6.5 mL of DMS
and 1 .O mL of p-cresol.
2. Place the reaction vessel into the HF apparatus mamfold. Evacuate the
system for l-2 min. Note that the DMS will begin to boil. Close the valve
to the reaction vessel, and cool with liquid nitrogen or dry ice-acetone.
3. Distill 2.5 mL of HF/g of resin. Allow the reaction vessel to warm up to
OOC. The p-cresol and p-thiocresol will slowly melt, freeing the star bar.
Cleave this mixture for 2 h at 0°C. At this point, peptides that do not con-
tain Asp(cHx), Glu(cHx), Cys(Bzl), Cys(Meb), or Arg(Tos) synthe-sized on a
chloromethyl-resin support will be completely cleaved and deprotected
by extending the cleavage time to 3 h.
4. Using the water aspirator, evaporate the HF and most of the DMS. This
usually takes l-l.5 h.
5. The reaction vessel is evacuated and cooled as before, and recharged
with HF. We generally charge the vessel with 10 mL of HF/g of resin, The
peptide resin is cleaved for l-l .5 h at -5 to 0°C.

14. Cleavage examples
Although no peptide is considered trivial, the cleavage procedure greatly
affects the overall yield of the desired product. The following examples
are provided to help in the selection of cleavage cocktails for a variety of
different peptides.
1.Amylin(Human)
2.Glu3,4,5,7,10,14-contoxin-G
3.ɑ-MSH Antagonist
4.Anthopleurin A

15. Amyl Sequence: H-Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-
Asn-Phe-Leu-Val-Hls-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser- Thr-
Asn-Val-Gly-Ser-Asn-Thr-Tyr-NH2
Characteristics: 37-mer, basic with amidated C-termmus; contains 1 mtra-
molecular disulfide bond; rather hydrophobic; considered difficult to
make.
Resin: Methylbenzhydrylamine (results in C-termmal amide).
Cleavage: 4.0 g resin with 4.5 mL anisole and 40 mL HF at 0°C for 60 min.
Extraction: Peptide dissolved in 400 mL of 8M urea containing O.lM Tris-
acetate, pH 7.2, followed by a ferricyanide oxidation procedure (24).
Remarks: Ammo actd analysis and FAB/MS (both after oxidation and
purification) in accordance with theory. in (human).

17. REFERENCE:
1.article method in molecular biology
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18. THANK YOU