Garner Group
Progress Report 1
12/11/15
Hans Cocks
Background
From the beginning of my tenure until now, my work has been directed towards two
main problems. The first concerning the synthesis of protected methyl-a-Dmannopyranoside carbohydrate derivatives for use in GPI related syntheses, and the
second involving the synthesis of cysteine thiol protecting groups for use in solution and
solid-phase peptide chemistry. Scheme 1 and 2, respectively, relate to the exclusive acyl
and benzyl protection of secondary alcohols contained in the methyl-a-D-mannopyranoside
sugar. The involved reactions served as a good introduction to the structural diversity of
natural sugars and are quite relevant considering the modification of natural
polysaccharides is gaining increased attention in the field of nano-medicine. Successful
preparation and preservation of stereochemistry in these protected sugars allows them to
be utilized in constructing sugar moieties which are ligated to other moieties in the
convergent synthesis of the CD52 GPI anchor found in leukocytes, epididymal cells, and
sperm. The primary unprotected alcohol site is for attachment of the next sugar, while the
protected alcohol groups are cleaved in the final global de-protection step of the GPI
synthesis. Schemes 3 and 4, respectively, outline the synthesis of base and acid labile
cysteine thiol protecting groups. These groups may be used in divergent orthogonal peptide
synthesis strategies and are preferred over other protecting groups because they enable
the prevention of racemization. Each group is, additionally, stable to removal of the other
under appropriate conditions, thus allowing for, in a strategy utilizing both groups, selective
de-protection of distinct cysteine thiol groups. Preparation of the base labile 9fluorenylmethylthiol molecule, shown in scheme 3, is, in use, comparable to other cysteine SH protecting groups such as the 2-(2,4dinitrophenyl)ethyl (Dnpe) group, though S-Fm
offers increased compatibility with other sulfur protecting groups. Preparation of acid labile
S-Tmob (2,4,6-trimethoxybenzylthiol) is ongoing; the protected group it introduces is
analogous to the p-methoxybenzyl (Mob) protecting group commonly used for the same
purpose, though S-Tmob has improved stability. Successful preparation of S-Tmob and SFm will allow for diversity, control, and stability in the synthesis of cysteine containing
peptides.
Results and Discussion
Scheme 1 - (reactions 1-3) Preparation of:
Methyl 2,3,4-tri-O-acetyl-alpha-D-mannopyranoside-6-ol
All reactions for scheme 1 proceeded well with little complications. Primary
alcohol A was treated with trityl chloride, pyridine was added to dissolve the
components and scavenge HCl, leading to formation of the trityl protected sugar B in
44% yield after purification via flash chromatography in 80% EA:Hex. Product was
compared against a previously prepared pure standard and characterized via H1 and
C13NMR which indicated high purity. Trityl-protected B was then acetylated with acetic
anhydride to protect the secondary alcohols leading to formation of the completely
protected alcohol C. Pyridine was again added to scavenge protons while serving as the
solvent. TLC in 10% EA:Hex indicated reaction had gone to completion and the mixture
was purified via flash chromatography in the same solvent system. The purified
product, obtained in 80% yield, was checked for purity against a previously prepared
standard in 40:60 EA:Hex and was characterized via H1 and C13NMR spectroscopy.
Protected sugar C was then de-tritylated by stirring with iron(III) chloride hexahydrate to
form the free primary alcohol D. TLC against a previously prepared pure std. in 40:60
EA:Hex indicated starting material to be in the crude mixture which was removed
through purification using flash chromatography eluting and running the column with
20:80 EA:Hex. The pure product D, was thus obtained in 75% yield and the product was
characterized via H1 and C13NMR spectroscopy.
Scheme 2 – (reactions 4-8) Preparation of:
Methyl 2,3,4-tri-O-benzyl-alpha-D-mannopyranoside-6-ol
The target product D of scheme 2 was never obtained due to an excess amount
of previously prepared material already existing. Methyl-alpha-D-mannopyranoside(A)
was treated under the same conditions as found in step 1, scheme 1 to produce the
trityl protected alcohol B in 58% yield(see exp. Trt-Man-4). Experiments 5 and 6
designated as 5-M-Trt-Bn and 6-M-DeTrt-Bn encountered complications and no usable
data was obtained. The trityl alcohol B was then successfully benzylated via addition of
NaH followed by BnBr leading to the fully protected intermediate C. After work-up the
obtained product was crystallized from CHCl3 adding hexanes to incipient turbidity
landing a final yield of 30%. Transition to scheme 3 occurred in the next reaction which
was a second preparation of the fully protected sugar C(Bn-Trt-M8), of which the
experiment was never completed. The fully protected sugar C was stored in a
dessicator and left for use at another time.
Scheme 3 – (reactions 9-15) Preparation of:
9-fluorenylmethylthiol
Table 1: Summary of reactions performed over course of scheme 3
Product
Attempted
Alcohol 2
Tosylate 3
Thioacetate 4
Target S-Fm 5
(Exp #) Trial 1, MP
(Exp #) Trial 2, MP
(Exp #) Trial 3, MP
(9F-OH-9) 25% yield,
MP = 95-97oC
(9H-Fm-Ts-10) failed
reaction
(9Fm-Sac-13) 35%
yield, MP=66-67oC
(9Fm-SH-15) 86%
yield, MP = 33-34oC
(9-Fm-OH-11) 25%
yield, MP = 82-85oC
(9Fm-Ts-12) 55%
yield, MP = 106-108
-
-
-
-
(9Fm-Ts-14) 44%
yield MP=105-107oC
-
Realizing the final product of scheme 3 proved to be difficult for a variety of
reasons. Small mistakes and lots of learning took place during this synthesis. Upon
successful isolation of each intermediate the products were characterized via melting
point and H1NMR spectroscopy. H1NMR inspection revealed all products to be
relatively pure. Below are excerpts, with the exception of 9F-OH-9 and 9Fm-Ts-10, from
prepared eBook entries regarding mechanisms and difficulties encountered during each
step of scheme 3:
Regarding formation of alcohol 2 (purified via recrystallization):
@9F-OH-9: Use of the word ligroin resulted in recrystallizing from a low boiling solvent,
the issue has since been reconciled, 50% recovery was achieved. MP of 95-97oC
indicates the product to be relatively pure.
@9Fm-OH-11: NaH deprotonates fluorene at the 9 position. The fluorene anion then
attacks the ethyl formate at the carbonyl carbon leading to loss of ethanol and the
desired aldehyde condensate intermediate. The aldehyde intermediate is then reduced
to a primary alcohol via addition of NaBH4. After working up the carbaldehyde
intermediate an attempt was made to ‘push’, based on the possibility of tautomerization
and hydration equilibriums, the yellow spot in the mixture to the desired aldehyde
product. Product was lost over several operations yielding no change in the presence of
the yellow spot, ending with reacidification of the mixture with GAA which was then
concentrated without an additional wash of sodium bicarbonate leading to the increased
usage of sodium borohydride mentioned above. After complete reduction and filtration,
the crude product, tinted slightly yellow, weighed 4.8086g (24.503mmol) corresponding
to a yield of 49.1%, with an M.P. of 83-87 C. TLC revealed 3 spots in the crude mixture.
Recrystallization resulted in the disappearance of 1-spot, and 50% recovery. A yellow
insoluble trapped on the bottom of the flask was set aside and the filtrate was saved for
if and when another attempt at harvesting crystals could take place. The MP for this
reaction was 16oC below that found in literature after recrystallization indicating a good
amount of impurities were trapped in the crystal.
Regarding formation of tosylate 3 (purified via incipient of turbidity using hexanes):
@9Fm-Ts-10: Excess pyridine was added to the reaction mixture, which after sitting
over a period of 3 days, lead to decomposition of the desired product. In retrospect, the
base lability of the Fluorenylmethyl group made this decomposition easy to predict.
@9Fm-Ts-12(14): The alcohol on compound 2 attacks the Sulfur atom of the paratoluene-sulfonyl chloride. The chlorine atom leaves grabbing the H+ ion at the original
alcohol site. Pyridine functions as an acid scavenger shifting equilibrium to the product.
Care should be taken not to let the reaction run to long, as excess pyridine may react
and deprotonate the 9H hydrogen ion leading to loss of the tosylate and formation of an
alkene by-product. Reactions 9Fm-Ts-12 and 9Fm-Ts-14 both proceeded smoothly with
no complications and good purity.
Regarding formation of thioacetate 4 (purified via flash chromatography in 1-4%EA:Hex)
:
@9Fm-Sac-13: The tosylate provides a good leaving group for substitution via
delocalization of the electrons it contains; the 18crown6 promotes the ability of the sulfur
atom to attack at the ch2 methyl site by chelating the potassium ion leading to
attachment and loss of the tosylate. This reaction is simple enough and should be high
yielding due to the reagents involved. I expect the next attempt at this reaction to
proceed with no problems. The majority of product loss for this reaction occurred due to
difficulty with getting good separation in the flash column leading to multiple purification
trials before separation was successful. The product was, in hind-sight, almost entirely
isolated during the first 3 columns but, due to researcher error, the product containing
fractions were mis-identified; future scenarios such as this may be prevented by rough
estimation of partition coefficients for compounds in the crude mixture via the vandeemter equation. The primary compound responsible for lack of proper separation (the
major impurity) was ‘a greasy mess’ which was isolated and characterized via HNMR.
Regarding formation of thiol 5, target molecule (purified via flash column in 2% EA:Hex):
@9Fm-SH-15: DIBAL-H, existing as a dimer and exhibiting electrophilic properties, is
first attacked by the carbonyl oxygen, it then gives its hydrogen to the carbonyl carbon,
forming a stable tetrahedral intermediate below -70oC. Excess (2.2 equiv.) of DIBAL-H
is used for this reaction, I hypothesize, due to the ability of the adjacent dimerized
aluminum to ligate to the nearby Sulphur atom after the attached DIBAL has given away
its hydrogen thus reforming a more stable dimer, and lowering the effective
concentration of DIBAL-H in the reaction mixture. Quenching of this reaction requires
two equivalents of HCl, one equivalent to give to the Sulphur atom thus creating a good
leaving group, and another to reform the original DIBAL-H dimer immediately prior to its
decomposition into the corresponding Aluminum oxide salt. Completion gave a high
yield but there is a slight amount of solvent in the product as seen in the HNMR
spectrum. However, melting point of the isolated product was within the bounds of that
found in literature indicating good purity. This reaction, though tricky, proceeded well
with no issues.
Scheme 4 – (reactions 16-19) Preparation of:
(2,4,6-trimethoxyphenyl)methanethiol
Scheme 4 is currently in progress; this project originally started after
complications arose in synthesizing the target acid labile cysteine protecting group
4b.Conversion of alcohol 3 to thiol 4b, via formation and hydrolysis of the thiuronium
intermediate salt 4a was attempted twice (see experiments Tmob-SH-16/17), with
consumption of the remaining in-house 2,4,6-trimethoxybenzyl alcohol before
developing the full scheme. The supplied literature (see references) indicated
quantitative yield to be achievable in high purity with no purification apart from simple
liquid-liquid extraction. Tmob-SH-16, the first attempt, resulted in a 52% yield, but the
product didn’t melt indicating all of it to likely be a salt. Notes on the reaction indicate:
the biggest deviation in this reaction occurred when water was lost during reflux.
Somehow the mixture became acidified and the product or byproducts became a salt. >> Future reaction should be done with excess and solid NaOH instead of a molar
solution. HNMR characterization of Tmob-SH-16 revealed lots of overlapping peaks at
3.5ppm with integrations way above expected. Tmob-SH-17, the second attempt, went
smoother achieving a 60% yield. Upon taking a melting point, much of the material
melted around 50oC (10oC below literature), but an un-melted portion still remained.
This was hypothesized to be the unreacted thiouronium salt which was reformed after
acidification of the mixture following hydrolysis of 4a. Upon preparing the NMR sample it
was noticed there was a small amount of insoluble material in the obtained product, the
sample was passed through a syringe filter before going into the NMR tube. HNMR
revealed the sample to be relatively pure thus at another time it may be possible to put
the thiol in solution and filter off the insoluble material to obtain the pure product. To
assist in peptide synthesis, the Stmob-SH-17 product was dissolved in dry ethyl acetate,
the insolubles filtered out using a syringe filter, and the mixture concentrated to near
dryness. Complete dryness, or the consistency expected, was unable to be achieved
and multiple High-Vac pumps were used to try and dry the product. Subsequently the
container with the product was evacuated, flushed with argon, tightly sealed with
parafilm and stored in the refrigerator. Two weeks later, the product was put back into
solution for an NMR attempt, and frustratingly, more of the insoluble material had
formed. This newly forming material could be the result of oxidation yielding the
disulfide. To prevent this decomposition in future reactions, lyophilizing the product
before storage could help dramatically. Other conditions are also being explored,
including using acetonitrile and tosic acid instead of water, acetone, and HCl. Use of
Cysteine residue stabilizers such as TCEP (tris(2-carboxyethyl)phosphine) is also being
considered. TCEP is a water soluble disulfide bond reducing agent, and is available
immobilized on agarose to assist in its removal upon reduction of the disulfide to the
free thiol. Starting from scratch, scheme 4 was initiated, (Stmob-ald-18) from compound
1, 1,3,5-trimethoxybenzene, to yield compound 2, 2,4,6-trimethoxybenzaldehyde in 74%
yield and high purity as indicated by H1NMR and MP analysis. The reaction proceeds
through the vilsheimer-haack reaction mechanism, first with formation of the vilsheimer
reagent, a chloroiminium salt of DMF and the added phosphorous(V) oxychloride, which
allows attachment of the aromatic ring thus proceeding to another iminium ion which is
hydrolyzed with water to form the desired benzaldehyde derivative. Sodium carbonate is
added to break down the phosphorodichloridic acid and HCl formed during the reaction.
No purification was needed, this simple, neat, reaction proceeded quickly and there
were no complications. Conversion of aldehyde 2 to alcohol 3 Stmob-OH-19 initially
proceeded well with complete formation of the alcohol taking place. Unfortunately the
product did not precipitate as expected (considering the pure standard is a white
crystalline material) and the mixture was not properly worked up in time. Over the days
the mixture sat on the bench, several decomposed variations of the target molecule
formed, to avoid this in the future, the mixture should be liquid-liquid extracted
immediately after decomposing the remaining hydrides with acetic acid. This issue
follows the same principles of decomposition, in terms of lability, as that which occurred
in the 9Fm-Ts-10 experiment. Scheme 4 will be revised and restarted from benzene
derivative 1, ideally with no more complications.
References and Notes
Carb. Res,, 179 (1988) 37-50
J. Org. Chem, 45(21), 1980. P. 4251
Gu, X. et al; Org. Lett. 2004, 6(19), 3285-3288, p. S4
Bandgar, B.P.; Totre, J. V. (Piramal Life Sciences Lt.). Cytokine Inhibitors. WIPO(PCT)
Patent, Publication Number: WO 2011/121505 A1. Application Number:
PCT/IB2011/051269. (Pg 44); Oct. 6, 2011
Crich, D; Kasinath, S; Guo ,S; Org. Lett. 2007, 9(22), 4423-4426, p.S3 p. S4.
Crich, D; Kasinath, S; Guo ,S; Org. Lett. 2007, 9(22), 4423-4426, p.S3 p. S3.
Synth. Comm., 28(17), 3219-3223(1998)